Anna Suchy Thesis

Development of processing technologies for
the generation of lignin with applications in

the production of high-value products
Anna SuchyAnna Suchy
Thesis submitted to Aberystwyth University for the degree of
Doctor of Philosophy in Biological Sciences
28.2.201828.2.2018
Supervisors
Dr. Ana Winters: Principal Investigator, IBERS, Aberystwyth University (alg@aber.ac.uk)
Dr. David Bryant: Synthetic Biologist, IBERS, Aberystwyth University (dgb@aber.ac.uk)
Dr. Lolke Sijtsma: Senior Scientist, Wageningen Food and Biobased Research,
(lolke.sijtsma@wur.nl)
i
THESIS WORD COUNT
70928
DECLARATION
This work has not previously been accepted in substance for any degree and is not being
concurrently submitted in candidature for any degree.
Signed...................................................................... (Candidate)
Date........................................................................
STATEMENT 1
This thesis is the result of my investigations, except where otherwise stated. Other
sources are acknowledged by footnotes giving explicit references. A bibliography is
appended.
Signed..................................................................... (Candidate)
Date........................................................................
STATEMENT 2
I hereby give consent for my thesis, if accepted, to be available for photocopying and
inter-library loan, and for the title and summary to be made available to outside
organisations.
Signed..................................................................... ( Candidate)
Date........................................................................
ii
ACKNOWLEDGEMENTS
I would like to begin by expressing my deepest gratitude to my main supervisor Dr Ana

Winters (Aberystwyth University) not only for her advice, support, patience and
knowledge which were essential to complete this work, but also for the valuable support
she offered during the last few years. Without her contribution most of what has been
achieved simply would not exist. I would also like to thank my second supervisors: Dr
David Bryant (Aberystwyth University) and Dr Lolke Sijtsma (Wageningen Food and
Biobased Research) for their help, guidance, confidence in my research and making me
think outside of the box when needed.
I also need to say thank you to all members of ADMIT Bio-Succinovate Project: Dr Richard
Gosselink, Dr Hans Mooibroek, Dr Screenivas Ravella, Dr Joe Gallagher, Dr Abhishek
Somani and Dr Steve Taylor for their assistance in my research and technical advice in
different areas. I am very grateful to other project researchers: David Walker and Eleni
Ioannou, not only for exchanging research experience but also for supporting each other
thourought the programme, trying to move the project further. Without the
determination of you all and hours spent in the lab, ADMIT Bio-SuccInovate would have
not gone that far. A special acknowledgment I would like to address to Sian Davies for
being a brilliant project manager and keeping us all together in all good and bad
moments. I am very privileged to have worked with you and extremely grateful to have
been given the opportunity to contribute to this project.
Additionally, I would like to thank Dr Barbara Hauck, Dr Noelia Villarroel Rodriguez, Dave
Thomas and of course Alberto Arnau for their valuable contribution in data analysis and
the completion of my lab work. I am also greatly indebted to Dr Naheed Kaderbhai for
all the guidance and help she provided during my studies.
This PhD would have not be completed without understanding, help and support of my
friends, especially during my long-term illness. A big thank you goes to Magda Dudek,
Maria Krolczyk, Jagoda Gieldon, Patrycja Brzdak (for hours spent on skype), Weronika
Wasiak, Kat Kowalska (for your endless optimism and positive energy), Gosia Raulyk,
Radek Dumanow (for your huge contribution in spring 2017), Pilar Marinez-Martin, Odin
Moron-Garcia, Stefani Dritsa (for having ‘coffee’’ when needed), Cristina Monterrubio-
iii
Martin, Dimitra Loka (for an endless care), Marta Malinowska, Evi Stavridou, Morgane
Eleouet, Magda Bylicka, Krzysztof Stefanczyk, Gina Garzon (for your huge empathy),
Joanna Wiliinska, Maciej Bisaga, Agnieszka Gladala- Kostarz (for your true honesty) and
Dorota Strzelecka (for simply being the best neighbour). It was very important to me to
be surrounded by such a dedicated and lovely people during my stay in Aberystwyth.
Mostly, I would like to thank Tom for putting up with me, for being patient and
supportive experiencing all ups and downs of my PhD. You are the best 
I am forever grateful to my family. The last 4 years were extremely difficult journey, and
I would not have been in the place I am now without you all.
Finally, I would like to express my gratitude to Climate-KIC for funding this studenship.
Thanks to the opportunities given to me I was able to establish international
collaborations and work with people who have been and will continue to play an
important role in my further career.
iv
SUMMARY
Lignin is one of the main components of lignocellulosic biomass and has been
traditionally regarded as a waste product of the paper and pulp industry. However, its
impressive chemical properties make it an ideal candidate feedstock for production of
bio-based materials. A challenge is that lignin is noted for its high recalcitrance towards
chemical and biochemical degradation processes. Moreover, the optimal lignin used for
biorefining should ideally have a low hemicellulose and sulphur content, be soluble in
organic solvents and show overall consistency in its properties. The hypothesis
addressed here is that combined steam explosion-organosolv and bioconversion
processes can be developed that will enable utilisation of the lignin components of
lignocellulosic feedstocks in an economically viable integrated biorefinery.
The aim of the research presented in this thesis was to identify a comprehensive
range of processing technologies for the generation of industrially useful forms of lignin.
Initial studies focused on methods of lignin extraction from grass (Miscanthus x
giganteus, wheat straw and corn stover) and wood (pine and willow), using the
formic/acetic acid/water organosolv method, with and without a steam explosion pre-
treatment. Generated fractions were then analysed in the context of composition and
suitability for the production of biomaterials. The results showed that processes, both
with and without pre-treatment, exhibit high lignin extraction efficiency for all types of
feedstocks, with the exception of pine. The organosolv process, combined with steam
explosion in acid conditions, significantly increased lignin extraction yield and was
optimal for Miscanthus giganteus. Comprehensive analysis carried out by wet chemical
analysis as well as FT-IR, 31P-NMR, thermogravimetric analysis (TGA) and size-exclusion
chromatography (SEC). revealed, that some of the generated lignin fractions have
properties suited to production of polyurethanes, polyesters and phenol-formaldehyde
resins.
In addition, the potential for microbial bioconversion of organosolv lignin into
chemicals and other bio-based products with commercial application was also
investigated. Development of new screening methods helped to establish that the fungal
species: Trichoderma harzianum, and the bacterial species: Pseudomonas putida,
Pelomonas saccharophila and Pseudomonas fluorescens, have the ability to modify and
degrade organosolv wheat straw lignin. Comprehensive study of lignin degradation
patterns showed, that the selected fungal and bacterial strains use fundamentally
different strategies for utilization of lignin. Identified break-down products indicated
cleavage of ester and β-O-4 ether linkages and catabolism of phenylcoumarans. Of the
four species, Pelomonas saccharophila showed the greatest potential for bioconversion
of organosolv lignin and production of high-value chemicals and building blocks for
polyesters, polycarbonates, epoxy and urethane resins. It was demonstrated, that
investigated strain produced both extracellular and intracellular laccase and it could be
a promising candidate for future use in biorefining.
The results presented in this thesis contributed to the development of processes for
extraction of lignin with properties suited to industrial applications. It also provided a
better understanding of organosolv wheat straw lignin structure and demonstrated
opportunities for microbial conversion into different types of bio-products. Ultimately,
it is envisaged that outcomes of this research will contribute to the development of the
European bioeconomy and lead to increased commercial utilization of lignin in the near
future.
v
TABLE OF CONTENTS
Thesis word count......................................................................................................... ii

Declaration .................................................................................................................... ii
Acknowledgements ..................................................................................................... iii
Summary ....................................................................................................................... v
Table of Contents......................................................................................................... vi
List of Figures ................................................................................................................ x
List of Tables.......................................................................................................... xivxiii
List of Abbreviations .............................................................................................. xixxiv
CHAPTER 1: GENERAL INTRODUCTION .............................................................................. 2518
1.1 Introduction .................................................................................................. 2518

1.2 The plant cell wall ......................................................................................... 2821
1.2.1 Plant cell wall biomass ................................................................................ 2821
1.2.2 Cellulose ....................................................................................................... 2922
1.2.3 Hemicellulose .............................................................................................. 3023
1.2.4 Lignin ............................................................................................................ 3124
1.3 Lignin isolation ............................................................................................. 3326
1.3.1 Native lignin- methods for the isolation of lignin in its
natural form (attempts of lignin isolation in the unchanged form) .................... 3427
1.3.2 Technical lignin- methods of isolation of lignin which alter the native form
3528
1.3.3 Lignin from biomass conversion processes for biorefining ....................... 3932
1.4 Applications of lignin in industry ................................................................. 4336
1.4.1 Near –term opportunities: power, fuel and syngas products ................... 4437
1.4.2 Medium-term opportunities: macromolecules ......................................... 4639
1.4.3 Long-term opportunities: low molecular weight aromatic
or phenolic compounds ......................................................................................... 4841
1.5 Valorisation of lignin .................................................................................... 5144
1.5.1 Current markets........................................................................................... 5144
1.5.2 Future markets ............................................................................................ 5245
1.6 Towards bio-based products- optimisation of lignin prescursors
vi
through physico-chemical and biological modification ...................................... 5447
1.6.1 Lignin modification and degradation by physico-chemical methods ....... 5548
1.6.2 Lignin modification/degradation by microorganisms and their enzymes 5951
1.7 Conclusions .................................................................................................. 6860
1.8 Aims of the study ......................................................................................... 6961
CHAPTER 2: EFFECT OF STEAM EXPLOSION ON THE EXTRACTION OF LIGNIN FROM GRASS AND WOOD 7163
2.1 Abstract ........................................................................................................ 7163

2.2 Introduction ................................................................................................. 7264
2.3 Materials and Methods ............................................................................... 7466
2.3.1 Feedstocks ................................................................................................... 7466
2.3.2 Steam explosion .......................................................................................... 7466
2.3.3 Lignin determination in starting material .................................................. 7567
2.3.4 Lignin Extraction .......................................................................................... 7667
2.3.5 Removing resins........................................................................................... 7769
3.3.6 Statistical analysis ........................................................................................ 7769
2.4 Results .......................................................................................................... 7870
2.4.1 Lignin content in starting material ............................................................. 7870
2.4.2 Lignin extraction .......................................................................................... 8072
2.4.3 Lignin extraction efficiency ......................................................................... 8576
2.4.4 Method development for lignin extraction from softwood ...................... 8880
2.5 Discussion ..................................................................................................... 8981
2.6 Conclusions .................................................................................................. 9586
CHAPTER 3: COMPARATIVE STUDY ON THE EFFECT OF PRE-TREATMENT WITH STEAM EXPLOSION ON
CHARACTERISTICS OF LIGNIN FROM GRASS AND WOOD........................................................... 9789
3.1 Abstract ........................................................................................................ 9789

3.2 Introduction ................................................................................................. 9891
3.3 Materials and Methods ............................................................................. 10194
3.3.1 Raw material .............................................................................................. 10194
3.3.2 Lignin analysis and characterisation ........................................................ 10194
3.4 Experiment I: Lignin characterisation from grasses ................................ 10497
3.4.1 Results ....................................................................................................... 10497
vii
3.4.2 Discussion ................................................................................................ 141134
3.5 Experiment II: Lignin characterisation from Wood ................................ 149142
3.5.1 Results ...................................................................................................... 149142
3.5.2 Discussion................................................................................................. 182175
3.6 Conclusions .............................................................................................. 190183
Chapter 4: Development of new methods for screening Organosolv lignin metabolising
organisms ........................................................................................................... 192185
4.1 Abstract .................................................................................................... 192185

4.2 Introduction ............................................................................................. 193186
4.3 Materials and Methods ........................................................................... 196189
4.3.1 Feedstocks ............................................................................................... 196189
4.3.2 Sources of microorganisms ..................................................................... 196189
4.3.3 Microbial culture development and maintenance ................................ 197189
4.3.4 Media for culturing and maintenance of microorganisms.................... 198190
4.3.5 Minimal media for screening of microorganisms and ligninolytic enzymes
199191
4.3.6 Specific media for primary screening of lignin-metabolising
fungi and bacteria ............................................................................................. 202194
4.3.7 Specific media for primary screening of ligninolytic enzymes .............. 202194
4.3.8 Characterisation of lignin metabolising fungi and bacteria .................. 203195
4.3.9 Enzyme screening assays ........................................................................ 205197
4.3.10 Laccase activity assay ............................................................................. 207199
4.3.11 Protein purification ................................................................................ 208200
4.4 Results ..................................................................................................... 209201
4.4.1 Primary screening of T. harzianum for growth on
a lignocellulosic feedstock and expression of ligninolytic enzymes capable
of metabolising lignin ........................................................................................ 209201
4.4.2 Primary screening of t. harzianum for growth on organosolv
lignin and expression of ligninolytic enzymes capable of metabolising lignin211203
4.4.3 Primary screening of bacterial strains for growth on organosolv
lignin and expression of ligninolytic enzymes capable of metabolising lignin217209
viii
4.5 Discussion ............................................................................................... 221213
4.6 Conclusions ............................................................................................. 227219
References.......................................................................................................... 314221
Appendix ............................................................................................................ 345222
ix
LIST OF FIGURES
Figure 1.1: Illustration of the cell wall structure (adapted from (Menon and Rao, 2012).
......................................................................................................................................... 2924
Figure 1.2: Cellobioside building unit in the cellulose polymer (Nagy, 2009). ............ 3025
Figure 1.3: The three monolignols- lignin precursors: (A) p-coumaryl, (B) coniferyl, (C)
sinapyl alcohols (Yinghuai et al., 2013.)......................................................................... 3227
Figure 1.4: Major linkages found in lignin polymers (adapted from Pandey and Kim, 2011).
......................................................................................................................................... 3328
Figure 1.5: Structural characteristics of Kraft pine lignin (Jongerius, 2013). ............... 3631
Figure 1.6: Structural characteristics of lignosulphonate (Jongerius, 2013). .............. 3732
Figure 1.7: Effects of pre-treatment on lignocellulosic biomass (Harmsen and Huijgen,
2010). .............................................................................................................................. 4136
Figure 1.8: Steam-exploded lignin obtained from steam explosion after enzymatic
hydrolysis (Zhang et al., 2011). ...................................................................................... 4136
Figure 1.9: Pyrolytic lignin obtained from thermochemical biomass conversion (Zhang et
al., 2011). ........................................................................................................................ 4237
Figure 1.10: Valuable products obtained from lignin (adapted from Yuan et al., 2013 ).
......................................................................................................................................... 4439
Figure 1.11: Main uses of Kraft lignin and lignosulphonate (NNFCC, 2009). ............... 5247
Figure 1.12: Lignin value vs lignin-based product value by lignin type (Gosselink, 2011).
......................................................................................................................................... 5449
Figure 1.13: Examples of main processes for lignin fragmentation (adapted from
Laurichesse and Avérous 2013). ................................................................................... 5550
Figure 1.14: Examples of the main routes for the synthesis of new chemical active sites
(adapted from Laurichesse and Avérous, 2013, Cateto et al. 2009). ........................ 5651
Figure 1.15: Examples of the chemical reactions for the functionalization of lignin
hydroxyl groups (adapted from Laurichesse and Avérous 2013). .............................. 5651
Figure 1.16: Schematic synthesis of lignin graft copolymers: a) grafting from, b) grafting
onto (Duval and Lawoko, 2014). .................................................................................... 5953
Figure 1.17: Lignin compound (I) and its degradation to guaiacol (II) and vanillic acid (III)
in the presence of bacteria (Buranov and Mazza, 2008).............................................. 6358
x
Figure 2.1: Steps involved in the extraction and recovery of lignin by the organosolv
process: 1) relflux condensor with round bottom flask in heating mantle containing
sample and extraction liquor, 2) lignin precipitation after addition of water, 3) lignin
pellet after centrifugation, 4) freeze-dried lignin extract............................................. 7771
Figure 2.2: Organosolv extracted lignin fractions from a range of non-pretreated
feedstocks: 1) Miscanthus giganteus (MG), 2) Corn stover (CS), 3) Wheat straw (WS), 4)
Willow (WW), 5) Pine (PN). ............................................................................................ 8175
Figure 2.3: Efficiency of lignin extraction from a rage of grass and wood feedstocks:
Miscanthus giganteus (MG), Wheat Straw (WS), Corn stover (CS), Pine (PN), Willow (WW)
following steam explosion of acid (SE Acid), alkaline (SE Alk) and non-pretretated
material. Data represent lignin yield as a percentage of total lignin content in starting
material with standard deviation (SD ±) of the mean (n=3). ........................................ 8680
Figure 3.1: 31 P-NMR spectra of CS SE Acid lignin ..................................................... 112107
Figure 3.2:Total content of functional groups as determined by 31 P-NMR in mmol g-1.
Standard deviation (SD) of the mean (n=2) is 0. ....................................................... 113108
Figure 3.3: Contents of functional groups in different lignin fractions. Data represent
aliphatic OH, cond.Phen.OH, syringyl OH, guaiacyl OH, p-Hydroxyl OH and COOH groups
in mmol g -1. Standard deviation (SD) of the mean (n=2) is 0. ............................ 114109
Figure 3.4: FTIR spectra of all extracted lignin fractions .......................................... 121116
Figure 3.5: FTIR spectra of lignin fractions extracted from pre-treated (SE Acid, SE Alk)
and non-pretreated material: A; WS, B; MG, C:CS ................................................... 122117
Figure 3.6: FTIR spectra of MG lignin fractions extracted from steam exploded (SE Acid,
SE Alk) and non-pretreated materials ....................................................................... 123118
Figure 3.7: FTIR spectra of WS lignin fractions extracted from steam exploded (SE Acid,
Figure 3.8: FTIR spectra of CS lignin fractions extracted from steam exploded (SE Acid,
Figure 3.9: FTIR spectra of MG SE Acid lignin (example) and lignin standards (CIMV, Kraft
lignin) ........................................................................................................................... 126121
Figure 3.10: Thermogravimetric (TG) curves of WS, CS and MG lignin fractions extracted
from steam exploded (SE Acid, SE Alk) and non-pretreated material .................... 129124
xi
Figure 3.11: Differential thermogravimetric (DTG) curves of WS, CS and MG lignin
fractions extracted from steam exploded (SE Acid, SE Alk) and non-pretreated material
..................................................................................................................................... 130125
Figure 3.12: Thermogravimetric (TG) and Differential thermogravimetric (DTG) curves
of WS, CS and MG lignin samples fractions from non-pretreated material ............ 131126
of WS, CS and MG lignin fractions extracted from steam exploded material with acid
treatment (SE Acid)..................................................................................................... 132127
of WS, CS and MG lignin fractions extracted from SE material with alkaline treatment (SE
Alk) ............................................................................................................................... 133128
Figure 3.15: Second derivative DTG curves of lignin fractions from different materials
(pre-treated with steam explosion and non-pretreated): A; non-pretreated, B; SE Acid, C;
SE Alk ........................................................................................................................... 134129
Figure 3.16: Size Exclusion Chromatography (SEC) profiles of grass lignin............. 137132
Figure 3.17: SEC profiles of WS SE Alk, WS SE Acid and WS lignin fractions compared to
lignin standards ........................................................................................................... 138133
Figure 3.18: SEC profiles of MG SE Alk, MG SE Acid and MG lignin fractions comparing to
lignin standards ........................................................................................................... 139134
Figure 3.19: SEC profiles of CS SE Alk, CS SE Acid and CS lignin fractions compared to lignin
standards ..................................................................................................................... 140135
Figure 3.20: 31 P-NMR spectra of PN SE Acid lignin. ................................................. 157152
Figure 3.21: Total contents of functional groups as determined by 31 P-NMR in mmol g-1.
Standard deviation (SD) of the mean (n=2) is 0. ....................................................... 158153
Figure 3.22: Contents of functional groups in different lignin fractions. Data represent
aliphatic OH, cond.Phen.OH, syringyl OH, guaiacyl OH, p-Hydroxyl OH and COOH groups
in mmol g-1). Standard deviation (SD) of the mean (n=2) is 0. ................................. 159154
Figure 3.23: FTIR spectra of all extracted lignin fractions. ........................................ 164159
Figure 3.24: FTIR spectra of lignin fractions extracted from treated (SE Acid, SE Alk) and
non-pretreated material: A; WW, B; PN.................................................................... 165160
Figure 3.25: FTIR spectra of WW lignin fractions extracted from steam exploded (SE Acid,
SE Alk) and non-pretreated materials. ...................................................................... 166161
xii
Figure 3.26: FTIR spectra of PN lignin fractions extracted from steam exploded (SE Acid,
SE Alk) and non-pretreated treated materials. ......................................................... 167162
Figure 3.27: FTIR spectra of PN SE Acid lignin (example) and lignin standards (CIMV, Kraft
lignin). .......................................................................................................................... 168163
Figure 3.28: Thermogravimetric (TG) curves of WW and PN lignin fractions extracted
from steam exploded (SE Acid, SE Alk) and non-pretreated material. ................... 171166
Figure 3.29: Differential thermogravimetric (DTG) curves of WW and PN lignin fractions
extracted from steam exploded (SE Acid, SE Alk) and non-pretreated material. ... 172167
Figure 3.30: Thermogravimetric (TG) and Differential thermogravimetric (DTG) curves of
WW and PN lignin fractions extracted from non-pretreated material.................... 173168
WW and PN lignin fractions extracted from steam exploded material with acid treatment
(SE Acid)....................................................................................................................... 174169
WW and PN lignin fractions extracted from SE material with alkaline treatment (SE Alk).
..................................................................................................................................... 175170
Figure 3.33: Second derivative DTG curves of lignin fractions extracted from different
materials (pre-treated with steam explosion and non-pretreated): A; non-pretreated, B;
SE Acid, C; SE Alk. ........................................................................................................ 176171
Figure 3.34: Size Exclusion Chromatography (SEC) profiles of wood lignin fractions.
..................................................................................................................................... 179174
Figure 3.35: SEC profiles of PN SE Alk, PN SE Acid and PN lignin fractions comparing to
lignin standards. .......................................................................................................... 180175
Figure 3.36: SEC profiles of WW SE Alk, WW SE Acid and WW lignin fractions comparing
to lignin standards. ..................................................................................................... 181176
Figure 4.1: Growth of T. harzianum on YNB solid media supplemented with different
amount of CIMV lignin after 5 days incubation at pH 5 and 28 oC. Test: A) YNB + lignin
0.25 % (w/v) + agar, B) YNB + lignin 0.5 % (w/v) + agar, C) YNB + lignin 1 % (w/v) + agar .
Control -: YNB + agar + inoculum, Control +: YNB + glucose 0.5 % (w/v) + agar + inoculum.
..................................................................................................................................... 212206
xiii
Figure 4.2: Growth of P.putida (1), P. saccharophila (2), P. fluorescens (3) plated on LB
plates after incubation in M9 liquid media supplemented with 0.25 % (w/v) of lignin for
96 hours at pH 7 and 30 oC (growth detected after 48 hours). ............................... 220214
LIST OF TABLES
Table 1.1: Overview of technology, product examples and challenges for conversion of
lignin to power-fuel-syngas products (adapted from Holladay et al., 2007). ............. 4645
Table 1.2: Overview of challenges for the use of lignin as a macromolecule (adapted from
Holladay et al., 2007). ..................................................................................................... 4847
Table 1.3: Overview of product examples and challenges for aromatics, including
benzene, toluene and xylene (BTX), phenols, monomeric lignin molecules and other low-
molecular weight products (adapted from Holladay et al., 2007). .............................. 5049
Table 1.4: Phenol and derivatives; production, market niche and application (Gosselink,
2011). .............................................................................................................................. 5150
Table 1.5: Organisms and enzymes involved in lignin conversion. .............................. 6665
Table 2.1: Lignin content in non-pretreated and steam exploded acid (SE Acid) and alkali
(SE Alk) treated plant feedstocks. Data represent Acid Insoluble Lignin (AIL), Acid Soluble
Lignin (ASL) and Total Lignin as a percentage of dry matter (DM) with standard deviation
(SD ±) of the mean (n=3). Values with the same letters within each lignin fraction are not
statistically different at p < 0.05 (based on Tukey’s comparison of means). .................. 79
Table 2.2: Organosolv lignin fractions extracted from range of grass feedstocks:
Miscanthus giganteus (MG), Wheat Straw (WS), Corn stover (CS) following steam
explosion of acid, alkali and non-pretreated material. Data represent lignin yield as a
percentage of dry matter (DM). ........................................................................................ 81
Table 2.3: Organosolv lignin fractions extracted from two wood feedstocks, Pine (PN)
and Willow (WW) following steam explosion of acid, alkali and non-pretreated material.
Data represent lignin yield as a percentage of dry matter (DM). .................................... 82
Table 2.4: Organosolv lignin fractions extracted from a range of grass and wood
feedstocks: Miscanthus giganteus (MG), Wheat Straw (WS), Corn stover (CS), Pine (PN),
Willow (WW) following steam explosion of acid (SE Acid), alkaline (SE Alk) and non-
xiv
pretreated material. Data represent lignin yield as a percentage of dry matter (DM) with
standard deviation (SD ±) of the mean (n=3). ................................................................... 84
Table 2.5: Efficiency of Organosolv lignin extraction process with a range of different
grass and wood feedstocks: Miscanthus giganteus (MG), Wheat Straw (WS), Corn stover
(CS), Pine (PN), Willow (WW) following steam explosion of acid (SE Acid), alkaline (SE Alk)
and non-pretreated material. Data represent lignin yield as a percentage of total lignin
content in starting material with standard deviation (SD ±) of the mean (n=3). Values with
the same letters are not statistically different at p < 0.05 (based on Tukey’s comparison
of means). ........................................................................................................................... 87
Table 2.6: Organosolv Pine (PN) lignin fractions extracted from extractives-free (EF) and
non-pretreated biomass. Data represent lignin yield as a percentage of dry matter (DM).
............................................................................................................................................. 88
Table 3.1: Analysis of lignin extracts from non-pretreated and acid or alkali steam
exploded Miscanthus giganteus, wheat straw and corn stover. Values are presented as a
percentage of dry matter (DM). Standard deviation (SD) of the mean (n=2) is 0. ND= not
detected. ..................................................................................................................... 106107
Table 3.2: Solubility of lignin extracts in different organic solvents. Values are presented
as a percentage of dry matter (DM) with standard deviation (SD ±) of the mean (n=3).
..................................................................................................................................... 108109
Table 3.3: Contents of functional groups as determined by 31P- NMR. The range of OH
groups include: aliphatic OH, cond.Phen.OH, syringyl OH, guaiacyl OH, p-Hydroxyl OH
and COOH groups (mmol g-1). Standard deviation (SD) of the mean (n=2) is 0. ND= not
detected. ..................................................................................................................... 111112
Table 3.4: Peak assignments for the FTIR spectra (Boeriu et al., 2004, El Hage et al., 2009,
Kline et al., 2010, Li et al., 2009, Monteil-Rivera et al., 2013). ................................ 119120
Table 3.5: S/G ratio based on FTIR method. .............................................................. 120121
Table 3.6: Proximate analysis of lignin samples determined from TGA graphs. Volatiles,
fixed carbon and ash are represented as a percentage of dry matter (DM). Standard
deviation of the mean (n=3) is 0. ............................................................................... 128129
Table 3.7: Average molar mass data (Mw, Mn) and polydispersity (Mw/Mn) of all lignin
samples. Standard deviation of the mean (n=2) is 0. ............................................... 136137
xv
Table 3.8: Analysis of lignin extracts from non-pretreated and acid and alkali steam
exploded pine and willow. Values are presented as a percentage of dry matter (DM).
Standard deviation (SD) of the mean (n=2) is 0. ND= not detected. ....................... 151153
Table 3.9: Solubility of lignin extracts in different organic solvents. Values are presented
as a percentage of dry matter (DM) with standard deviation (SD ±) of the mean (n=3).
..................................................................................................................................... 153155
groups include: aliphatic OH, cond.Phen.OH, syringyl OH, guaiacyll OH, p-Hydroxyl OH
and COOH in (mmol g-1). Standard deviation (SD) of the mean (n=2) is 0. ND= not
detected. ..................................................................................................................... 156158
Table 3.11: S/G ratio based on FTIR method. ............................................................ 163165
Table 3.12: Proximate analysis of lignin samples determined from TGA graphs. Data
represent max temperature (Tmax) as well as volatiles, fixed carbon and ash as
percentage of dry matter (DM). Standard deviation of the mean (n=3) is 0. ........ 170172
Table 3.13: Average molar mass data (Mw, Mn) and polydispersity (Mw/Mn) of all lignin
samples. Standard deviation of the mean (n=3) is 0. ............................................... 178181
Table 4.1: Composition of YPD media........................................................................ 198201
Table 4.2: Composition of LB media. ......................................................................... 198201
Table 4.3: Composition of YNB media. ...................................................................... 200203
Table 4.4: Composition of M9 media (minimal salts). .............................................. 201204
Table 4.5: Growth of T. harzianum in YNB liquid media supplemented with different
amounts of Miscanthus giganteus (MG) on a weight per volume basis (w/v), at pH 5 and
28 oC. Control – : YNB liquid media and inoculum, Control +: YNB liquid media with 0.5
% (w/v) of glucose and inoculum. .............................................................................. 210213
Table 4.6: Laccase activity of extracellular enzymes produced by T. harzianum in YNB
liquid media with different amounts of Miscanthus giganteus (MG) on a weight per
volume basis (w/v), determined after 30 minutes at pH 5, pH 7 and 30 oC. Control-: no
enzyme, Control + laccase from T. versicolor............................................................ 211214
Table 4.7: Dye decolourisation during growth of T. harzianum on YNB solid media
supplemented with methylene blue (MB) and different amounts of lignin on a weight per
volume basis (w/v), at pH 5 and pH 7 incubated at 28 oC for 5 days. Control-: uninoculated
plate, Control +: plate supplemented with laccase from T. versicolor. ................... 214217
xvi
Table 4.8: Growth of T. harzianum after 10 days growth in YNB liquid media
supplemented with different amounts of lignin on a weight per volume basis (w/v), at pH
5 and 28 oC. Control -: YNB liquid media and inoculum, Control +: YNB liquid media with
0.5 % (w/v) of glucose and inoculum......................................................................... 215218
Table 4.9: Growth of T. harzianum in YNB liquid media supplemented with different
amounts of lignin on a weight per volume basis (w/v) at pH 7 incubated at 28 oC for 10
days. Control -: YNB liquid media and inoculum, Control +: YNB liquid media with 0.5 %
(w/v) of glucose and inoculum................................................................................... 216219
Table 4.10: Dye decolourisation in spent culture supernatants after incubation of T.
harzianum in YNB liquid media with different amounts of lignin on a weight per volume
(w/v) basis at pH 5 or pH 7, and incubated at 28 oC for 10 days. Control-: no enzyme,
Control +: laccase from T. versicolor.......................................................................... 217220
Table 4.11: Growth of P. putida, P. saccharophila and P. fluorescens after 10 days
incubation on M9 solid media supplemented with 0.25 % (w/v ) of lignin at pH 7 and 30
oC. Control -: M9 solid media and inoculum, Control +: M9 solid media with 0.25 % (w/v)
of glucose and inoculum. ........................................................................................... 218221
Table 4.12: Dye decolourisation during growth of P. putida, P. saccharophila, P.
fluorescens on M9 solid media supplemented with methylene blue, glucose and lignin
on a weight per volume basis. for 10 days at pH 7 and 30 oC.Control-: uninoculated plate,
Control +: plate supplemented with laccase from T. versicolor............................... 219222
Table 5.1: Growth of T. harzianum in YNB liquid media supplemented with 0.25 % (w/v)
of CIMV lignin at 30 oC . Control -: YNB liquid media and inoculum, Control +: YNB liquid
media with 0.25 % (w/v) of CIMV lignin, 0.25 % (w/v) of glucose and inoculum.... 239243
Table 5.2: Lignin break-down products and lignin-related phenolics identified in cell-free
extracts after incubation of P. putida, P. saccharophila, P. fluorescens and T. harzianum
in M9 liquid media supplemented with 0.25 % (w/v) of lignin for 360 hours at 30 oC.
..................................................................................................................................... 243247
Table 6.1: Solutions used for preparation of a mini 12 % SDS-polyacrylamide gel. 286290
Table 6.2: Average concentration of proteins extracted at different time points with
standard deviation (SD±) of the mean (n=2). ................................. 289293
Table 6.3: Laccase activity of extracellular and intracellular enzymes extracted of P.
saccharophila grown in M9 liquid media with 0.25 % (w/v) of CIMV lignin determined
xvii
visually and spectophotometrically after 14 hours incubation at pH 5, pH 7 and 30 oC.
Control -: no enzyme, Control +: laccase from T. versicolor. ................................... 291295
Table 6.4: Laccase activity of extracellular and intracellular enzymes produced by P
.saccharophila in M9 liquid media with 0.25 % (w/v) of CIMV lignin determined visually
at pH 6 and 30 oC in control and test experiment. Control: - no enzyme, Control +: laccase
from T. versicolor. ....................................................................................................... 293297
Table 6.5: Average molar mass data (Mw, Mn) and polydispersity (Mw/Mn) of lignin
samples modified by P. saccharophila in M9 liquid media. Standard deviation (SD) of the
mean (n=2) is 0. .......................................................................................................... 294298
groups include: aliphatic OH, cond.Phen.OH, syringyl OH, guaiacyl OH, p-Hydroxyl OH
and COOH (mmol g-1). Standard deviation (SD) of the mean (n=2) is 0. ND= not detected.
..................................................................................................................................... 296300
xviii
LIST OF ABBREVIATIONS
A280 Absorbance measured at 280 nm

AAD Aryl-alcohol dehydrogenase
AAO Aryl-alcohol oxidase
ABTS 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid
ADH Aryl alcohol dehydrogenase
AIL Acid Insoluble Lignin
ANOVA Analysis of Variance
APS Ammonium persulphate
ASL Acid Soluble Lignin
ATR Attenuated Total Reflectance
AU Aberystwyth University
BSA Bovine Serum Albumine
BTX Benzene, Toluene and Xylene
°C Celsius
C Carbon
C- Negative control
C+ Positive control
C-C Carbon-Carbon
CaCL2 Calcium chloride
Ca(OH)2 Calcium hydroxide
C-H Carbon-Hydrogen
C-O Carbon-Oxygen
CDCl3 Deuterated chloroform
CDH Cellebiose dehyrogenase
CH4 Methane
CIMV Compagnie Industrielle de la Matière végétale
cm Centimetre
CO Carbon monoxide
CO2 Carbon dioxide
xix
Cond.Phen Condensed phenolic
CS Corn stover
CuCl2 Copper chloride
CuSO4 Copper sulfate
DM Dry matter
DME Methanol/Dimethyl ether process
DMF Dimethylformamide
DMSO Dimethyl sulfoxide
DTG Differential thermogravimetric
DypB Manganese-dependent lignin peroxidase
e.g. For example
FT Fischer-Tropsh technology
FT-IR Fourier Transform Infrared Spectoscropy
g Gram
G Guaiacyl
GHP 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone
GLOX Glyoxal oxidase
G$ Guyana Dollar
GST Glutathione-S-transferase
GT Growth detected
Gt Gigatonne
H p-hydroxyphenyl
H2 Hydrogen
hm3 Cubic hectometer
H2O2 Hydrogen peroxide
HPAEC-PAD High performance anion exchange chromatography coupled to
pulsedamperometric detection
H3PO4 Phosphoric acid
H2SO4 Sulfuric acid
IBERS Institute of Biological, Environmental and Rural Sciences
kDa Kilodalton
kV Kilovolt
LB Luria-Bertani
LC-PDA- Liquid Chromatography-Photodiode Array Detection-Tandem Mass
xx
ESI/MSn Spectometry
LiP Lignin peroxidase
LPCA Lignin-based polycarboxylic acid
LPM Lignin Precipitation System
M Molar
MB Methylene blue
MG Miscanthus giganteus
mg Milligram
MgCl2 Magnesium chloride
MgSO4 Magnesium sulfate
M-H Negative ion mode
min Minute
ML Milled
mL Millilitre
mm Millimeter
mM Millimolar
Mmol g-1 Millimole per gram
Mn Number average molecular weight

MnP Manganese peroxidase
Mt Megaton
MTG Methanol to gasoline
MTO Methanol to olefin
Mw Weight average molecular weight
MWL Milled wood lignin
m/z Mass to charge ratio
M9 Minimal salts
N2 Nitrogen
NaOH Sodium hydroxide
ND Not detected
NM Not milled
NoP Novel peroxidase
NREL National Renewable Energy Laboratory
OD590 Optical density at 590 nm
OH Hydroxyl
xxi
PCBER Phenylcoumaran benzylic ether reductase
PD Polydispersity
PF Pseudomonas fluorescens
PF resins Phenol-formaldehyde resins
PMSF Phenylmethylsulfonylfluoride
PN Pine
31P-NMR Phosphorus-31 Nuclear Magnetic Resonance
PP Pseudomonas putida
PS Pelomonas saccharophila
PU Polyurethane
QR Quinone reductase
RGB Running gel buffer
ROP Ring Opening Polymerisation
rpm Revolutions per minute
RT Retention time
S Syringyl
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SE Acid Steam explosion with acid treatment
SE Alk Steam explosion with alkaline treatment
SEC Size Exclusion Chromatography
S/G Syringyl/Guaiacyl
SGB Stacking gel buffer
SHP 3-hydroxy-1(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone
SPE Solid Phase Extraction
STDEV Standard deviation
TBD Triazabicyclodecene
TEMED Tetramethylethylenediamine
Temp Temperature
TGA Thermogravimetric analysis
TH Trichoderma harzianum
Tmax Temperature maximum
UV Ultraviolet
VP Versatile peroxidase
v/v Volume per volume
xxii
w/v Weight per volume
WS Wheat straw
WW Willow
YNB Yeast Nitrogen Base
YPD Yeast Peptone Dextrose
µg Microgram
µM Micromolar
µl Microlitre
µM Micrometre
µm Micromol
xxiii
Chapter 1
24
Chapter 1: GENERAL INTRODUCTION
1.1 INTRODUCTION
There is currently a high demand for consumer products such as plastics, transportation
fuels, materials and chemicals. These products, mainly originate from fossil resources,
which will be depleted sooner or later (Gosselink, 2011). Moreover, use of fossil
resources is associated with many global problems such as environmental pollution,
climate change or security of supply (de Wild, et al., 2012).
Awareness, that utilization of fossil raw material contributes to CO2 emissions and the
''greenhouse effect’’ has led to strong interest in the use of renewable biomass resources
and sustainable development (Nagy, 2009). Biomass as a renewable and sustainable
resource is the only alternative to replace carbon from fossil sources for the production
of carbon-based products such as, chemicals, materials and liquid fuels (Kazmi, 2012).
In order to limit the green-house effect, several governments have adopted policy to
increase in domestic energy and chemical production from renewable resources,
especially biomass. The U.S. Department of Agriculture and U.S. Department of Energy Commented [AW[1]: Changed wording in case too much like
original
have set a target to obtain 20 % of transportation fuels and 25 % of U.S. chemical
commodities from biomass by 2030 (Jongerius, 2013). Similarly, in Europe, the Dutch Commented [AS[2]: check
Ministry of Economic Affairs set a goal to derive 30 % of transportation fuels from

biomass and to substitute 20 %-45 % of fossil-based raw materials by biomass by 2040.
The European Union member states are obliged to provide 20 % of total energy
consumed in the form of renewable energy by 2020. These goals have contributed to
development of technology and processes for biomass valorisation (Zakzeski et al.,
2010). Such processing of biomass to value-added products and energy can take place in
biorefineries (Morais and Bogel-Lukasik, 2013).
The main aim of a biorefinery is to develop processes which utilise by-products (including
agricultural waste) for use as a local source of power, biogas or for transformation into
added-value biomaterials, bio-based chemicals or biofuels (Fava et al., 2013). Although,
all kinds of biomass can be theoretically converted into fuels and chemicals, the supply
of edible crops is becoming a big problem in the world with the current population
25
density. In order to reduce competition with the use of land for the production of food,
it is recommended to exploit second generation feedstocks, for example lignocellulosic
biomass which grows on more marginal land (Naik et al., 2010).
Lignocellulosic biomass, including energy crops and agricultural and wood waste is a
renewable resource with significant potential (Zeng et al., 2014). These types of feed
stock have advantages in terms of chemical composition, plentiful availability and
relative low cost of conversion into products and offer many possibilities for the energy
and industry sector (Gosselink, 2011). Lignocellulosic biomass is composed of three
major components: cellulose, hemicellulose and lignin (Yuan, 2013). The cellulose and
hemicellulose fractions, which can both be hydrolysed to monomeric sugars, are
commonly used in the production of biofuels, platform chemicals, and synthesis
surfactants or polymers (Gosselink et al., 2011). Older biorefineries based on plant
biomass were mainly based on use of easily convertible fractions (Pandey and Kim,
2011). Lignin is a highly complex phenolic polymer which has been historically considered
as a 'waste product' in paper and biofuel production (Lange et al., 2013). Large amounts
of lignin are produced every year by the paper and pulp industry, as a by-product of
delignification (El Hage et al., 2009). However, the chemical structure of lignin indicates
that it might be a potential source of high-value products (Cotana et al., 2014).
Increased production of second-generation biofuels using lignocellulosic biorefinery

processes will result in the production of a large amount of lignin (Gosselink et al., 2012).
To meet the target of replacing 30 % of fossil fuels by biofuels by 2030, it is estimated
that t 227 hm3 of bioethanol would be required, which would generate about 0.225 Gt
of lignin (Sahoo et al., 2011).
Lignin is the most abundant aromatic polymer and second most abundant organic
polymer on earth (after cellulose) (Leisola et al., 2012). Direct use of lignin or conversion
to lower-molecular weight aromatics and building blocks for polymer production can
create a commercial opportunity for the future (Lange et al., 2013). The most promising
high value applications of polymeric lignin include carbon fibres, polymer modifiers (for
example utilising lignin as high value copolymer additives) or adhesives and resins (for
example for formaldehyde-free applications) (Zakzeski et al., 2010). In turn, aromatic
building blocks derived from lignin can be further converted into a variety of useful
26
chemicals. Lignin-derived chemicals are also a potential major source of raw material for
plastics, resins and other polymer materials (Jongerius, 2013).
Market demands for aromatic and phenolic compounds are currently enormous (Zhang
et al., 2011). Global annual production of phenol, used mainly for manufacturing of bis-
phenol A in polycarbonate production and for PF-resins is about 8 million tons (Gosselink
et al., 2012). Several studies have already been carried out on the conversion of lignin to
value-added products. The problem is, that lignin is very difficult to degrade and
generates very high amounts of solid residue compared to other fractions of
lignocelluloses (Pandey and Kim, 2011). Therefore, a myth in industry which states ‘You
can make anything out of lignin, except money’ is still a challenge (Holladay et al., 2007).
A major effort is needed to develop new technology for optimisation of lignin value
creation (Zakzeski et al., 2010).
The main aim of this chapter is to describe lignocellulosic biomass, focusing mainly on
lignin and our current knowledge regarding different techniques of lignin isolation and
conversion to high-value products. This will help to identify strategies to overcome
technical barriers and potential technologies which can make lignin an economically
feasible resource.
27
1.2 THE PLANT CELL WALL
1.2.1 PLANT CELL WALL BIOMASS
Plant biomass is one of the greatest, untapped reserves on the planet. It is mostly
comprised of cell walls which are largley made up of polymers including cellulose,
hemicellulose and polyphenolic lignin. Therefore, plant materials with a high content of
cell wall components are generally called lignocellulosics (Figure 1.1) (Foster et al., 2010).
Lignocellulosic biomass represents one of the world’s largest renewable biochemical

resources, with 77 x 10 9 metric tons produced annually. It can provide a sustainable
feedstock for several biorefinery technologies (Nagy, 2009).
Lignocellulose consists mainly of cellulose, hemicellulose and lignin. However, some

inorganic materials and other extractives are observed as well (Berg, 2013). Cellulose
content of lignocellulose varies from about 35 % – 50 %, the hemicellulose content from
about 20 % - 35 % and the lignin content from about 10 % – 25% (Ong, 1996). Cellulose
and hemicelluloses are tightly linked to the lignin component, through covalent and
hydrogen bonds (Limayem and Ricke, 2012). Basically, the cellulose component forms a
scaffold which is surrounded by other components which function as a matrix
(hemicelluloses) and binding (lignin) materials (Adhikari and Satyanarayana, 2007).
Lignocellulosic biomass can be provided in two forms: as a crop or as a residue.

Enormous amounts of cellulosic biomass are usually produced via dedicated crops like
perennial herbaceous plant species or short rotation woody species. Other sources of
lignocellulosic biomass are waste and residues, such as straw from agriculture, or wood
waste from the pulp and paper industry and forestry residues (Cherubini, 2010). More
recently, there is also growing interest in using organic municipal waste and marine algae
as a source of lignocellulosic biomass (Limayem and Ricke, 2012).
28
Figure 4.1: Illustration of the cell wall structure (adapted from (Menon and Rao, 2012).
1.2.2 CELLULOSE
Cellulose is the main component of the plant cell wall, playing a key role in structural
support for cells (Agbor et al. 2011). As the most abundant polymer on earth, it can also
be present in algae, fungi and bacteria (Siqueira et al., 2010). Cellulose, is a linear
homopolymer of (1-4)-linked β -D-glucopyranosyl units with a degree of polymerization
(DP) ranging from a couple of hundred up to several thousand (Figure 1.2) (Nagy, 2009).
The long-chain cellulose polymers are linked together by hydrogen and van der Waals
bonds, which pack the cellulose into microfibrils (Kumar et al., 2009). Cellulose can be
present in several different supra-molecular structures, including amphorous, or
crystalline forms (Nagy, 2009).
Crystalline cellulose comprises the major part of cellulose whereas only a small
percentage of unorganized cellulose chains forms amorphous cellulose. Cellulose in its
amorphous form is more susceptible to enzymatic degradation (Kumar et al., 2009).
Native cellulose has been shown to be a composite mixture of two crystalline
allomorphs, designated as Iα and Iβ. Cellulose Iα is dominant in bacterial and algal
cellulose, whereas Iβ crystalline form can be seen in higher plants (Rudsander, 2007). It
is worth mentioning, that Larsson et al., (1997) have also proposed the occurrence of a
para-crystalline form of cellulose which is less ordered than crystalline Iα and Iβ
allomorphs, but more ordered than amorphous domains. These differences in crystalline
29
packing forces and order have a wide impact on a variety of chemical and enzymatic
reactions with cellulose and can affect/need to be considered in future industrial
applications (Nagy, 2009).
Figure 4.2: Cellobioside building unit in the cellulose polymer (Nagy, 2009).
1.2.3 HEMICELLULOSE
The term hemicellulose is usually used to describe a family of polysaccharides and their
derivatives that are found in the plant cell wall and vary in composition and structure
depending on their source and the method of extraction (Harmsen and Huijgen, 2010).
Hemicelluloses have an intermediate degree of complexity (Martínez et al., 2005).
Typical hemicellulose is an amorphous and variable structure, built of heteropolymers
including hexoses (D-glucose, D-galactose, D-mannose), as well as pentoses (D-xylose, L-
arabinose) and sugar acids i.e. D-glucuronic, D-galacturonic and methylgalacturonic acids
(Limayem and Ricke, 2012). Hemicellulose backbones are typically β-1,4-linked polymers
of xylopyranose and glucose which can be decorated with substituent groups that
prevent crystalline formation and promote solubility, thereby maintain flexibility and
distance between cellulose microfibrils (Phitsuwan et al., 2013). Branching frequency can
vary depending on the nature and the source of feedstocks (Limayem and Ricke, 2012).
The principal component of hardwood hemicellulose is glucuronoxylan, whereas
glucomannan can be seen in softwood (Pérez et al., 2002). Moreover, the degree of
substitution and types of substituent groups vary with plant species too. For example,
xylan in hardwood is highly acetylated and branched with small amounts of glucuronic
acids, whereas in softwood, it is partially acetylated and mainly branched with arabinose
and uronic acids (Phitsuwan et al., 2013). In grasses, the xylan backbone is typically
decorated with arabinose residues which are frequently linked with ferulic acid. Arabino-
30
xylans can link with lignin through feruloyl residues (Marcia, 2009). Compared to
cellulose, hemicelluloses are branched with short lateral chains consisting (Marcia, 2009)
of different sugars. They are easily hydrolysable polymers and which do not form
aggregates, even when they are co-crystalized with cellulose chains (Pérez et al., 2002).
1.2.4 LIGNIN
1.2.4.1 General properties of lignin
The term lignin is derived from the Latin word lignum meaning wood (Chakar and
Ragauskas, 2004). It is a natural polyaromatic and amorphous polymer, acting as the glue
that gives plants their structural integrity (Jongerius, 2013). Moreover, through
crosslinking with cellulose and hemicellulose lignin also helps in water transport and
protection against attacks by insects and microorganisms (Holladay et al., 2007). As an
extremely abundant raw material, it contributes as much as 30 % of the weight and 40
% of the energy content of lignocellulosic biomass (Jong, 2011). There are two different
types of lignin: native lignin, and technical lignin. Native lignin is present in biomass
(Holladay et al., 2007). It can be divided into three classes which are commonly called
softwood (gymnosperm), hardwood (dicotyledonous angiosperm) and grass or annual
plant (monocotyledonous angiosperm) lignins (Pearl, 1967). Technical lignin is a form of
lignin obtained following isolation from biomass through different processes (Holladay
et al., 2007). Details about different types and availability are described in section 1.3.
1.2.4.2 Lignin structure

Lignin is the most common complex biopolymer (Phitsuwan et al., 2013). It consists of p-
hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units (Kang et al., 2013). Nature produces
lignin by dehydrogenative polymerisation of three monolignols, namely: p-coumaryl,
coniferyl and sinapyl alcohol (Figure 1.3) (Amen-Chen et al., 2001). Initially, two plant
enzymes (peroxidase and laccase) cause the dehydrogenation of two phenolic OH groups
and generate intermediate radicals from lignin precursors. The polymerization process
starts by random phenol radical-radical coupling reactions, creating dimers, then trimers,
oligomers and finally complex branched polymer (Pandey and Kim, 2011).
Lignin is a three dimensional network, lacking regular and ordered repeating units
(Gosselink, 2011). It is hydrophobic and acts like a coating around the cellulose fibrils
31
(Nagy, 2009). Composition of lignin varies from plant to plant (Mattinen et al., 2008). In
softwood species, lignin mainly consists of guaiacyl units, while lignin from hardwood
species consists of both guaiacyl and syringyl units. All three monolignols are observed
in lignin from herbaceous plants (de Wild et al., 2012).
It is worth mentioning, that lignin contains a range of functional, chemical groups. The
main groups in native lignin are hydroxyl, methoxyl, carbonyl and carboxyl (Boeriu et al.,
2004). The monomeric units of phenylpropane in lignin polymers are linked in a complex
network through different types of ether, ester as well as carbon-carbon bonds (Zhang
et al., 2011).
There are several different types of linkages and they include β-O-4, 5-5, β-5, 4-O-5, β-1,
α-O-4 and β-β linkages (Figure 1. 4) (Pandey and Kim, 2011). The numbering system,
based on lignin nomenclature rules is shown in Figure 1.3 A (Yinghuai et al., 2013). The
linkage β-O-4 is the most dominant, comprising more than half of the linkage structure
of lignin in both, softwood and hardwood species (Zakzeski et al., 2010) making up
approximately 50 % of spruce linkages and about 60 % of birch and eucalyptus linkages
(Jongerius, 2013). Understanding these linkages plays a key role when considering
chemistries, products and technical barriers to creating high-value products from lignin
(Holladay et al., 2007). It is worth noting that coumaric acid is found extensively esterified
with lignin in grasses (Ralph et al., 1994).
Figure 4.3: The three monolignols- lignin precursors: (A) p-coumaryl, (B) coniferyl, (C) sinapyl alcohols
(Yinghuai et al., 2013.)
32
Figure 4.4: Major linkages found in lignin polymers (adapted from Pandey and Kim, 2011).
1.2.4.3 Physical properties of lignin

In terms of physical properties, natural lignin can be considered a thermoplastic. It
softens at 80 oC -120 oC and liquefies at 140 oC -145 oC (Kronkright, 1990). The glass
transition temperature Tg, which is an indirect measure of crystallinity and degree of
crosslinking and directly indicates conversion to a ‘rubbery’ state, is dependent on the
water and polysaccharide content, molecular weight and chemical functionalization of
the lignin. The molecular mass of lignin also depends on different factors (for example
method of isolation) and ranges from 1,000 g/mol to 20,000 g/mol (Doherty et al., 2011).
1.3 LIGNIN ISOLATION
Theoretically, lignin can be isolated by different extraction methods (Zabaleta, 2012).

However, the isolation of lignin with high yields and minimal chemical modification is still
a huge challenge (Kang et al., 2013). Despite the fact that a ''gold standard'' procedure
for lignin isolation does not exist yet, the isolation method plays a key role in determining
the nature and structure of lignin (Lange et al., 2013). Therefore, the most popular
isolation processes and characteristics of the obtained lignin extracts are presented
below.
33
1.3.1 NATIVE LIGNIN- METHODS FOR THE ISOLATION OF LIGNIN IN ITS NATURAL FORM (ATTEMPTS OF LIGNIN
ISOLATION IN THE UNCHANGED FORM)
1.3.1.1 Braun’s lignin

This method was first described in 1939 (Sakakibara et al., 2009). According to Brauns’
procedure, wood is first extracted with cold water and then with ether to remove
extraneous components. Subsequent extraction of a portion of the lignin with ethanol,
followed by purification steps gives ''native lignin'' (Kirk and Obst, 1988). Braun’s lignin
has been already characterized in 1971 by Lai and Sarkanen. They discovered that lignin
has a low molecular weight, high phenolic content and there are some extractives
associated with that. Also, the yield of isolated lignin is not large. ''Native lignin'' obtained
by this process represents only a small percentage of the total lignin in the wood (Pearl,
1967).
1.3.1.2 Milled wood lignin

The first description of the isolation of milled wood lignin (MWL) was published by
Björkman in 1956. According to this method, lignin is extracted from wood with a neutral
organic solvent (e.g. 1,4-dioxane) (Lange et al., 2013). The cell wall structure is disrupted
by grinding with a ball mill in the presence of an organic solvent such as water-dioxane
mixture and the dissolved lignin is purified by solvent precipitation. The final lignin
product can be obtained as an almost white powder (Jingjing, 2011). Regarding to
morphological origin of MWL, there are opposite opinions, based on different
fractionation and analysis methods (Guo et al., 2011). According to Lee et al., (1981),
MWL of earlier milling time is rich in the guaiacyl unit and MWL of longer milling time
results in a gradual increase of syringyl unit. Ozonation results, obtained by Akiyama et
al., (2005) show, that MWL is rich in a fraction derived from compound middle lamella,
at least at the initial stage of milling. The Björkman procedure is currently the most
common procedure for the isolation of lignin from wood. However, lots of investigators
still have not accepted it as a material truly representative the protolignin in wood (Guo
et al., 2011). Since MWL is soluble in several neutral solvents, the milling process
probably changes the lignin to some extent (Pearl, 1967).
34
1.3.2 TECHNICAL LIGNIN- METHODS OF ISOLATION OF LIGNIN WHICH ALTER THE NATIVE FORM
1.3.2.1 Lignin from pulping industry
The pulping industry is currently one of the greatest potential sources of isolated lignin
for industrial purposes. Pulp can be obtained from both wood and non-wood materials
by a range of different pulping processes. Different pulping methods result in the
removal of different amounts of lignin from pulp and therefore impinge upon the
amount of lignin which can be recovered for downstream uses (NNFCC, 2009). Different
pulping processes and lignin fractions which can be obtained from them are described
below.
Kraft lignin
Kraft pulping is the most dominant chemical pulping process in the world. It can be
conducted in the presence of substantial amounts of aqueous sulphite, sulfhydryl and
polysulphide, at high pH and at temperature between 150 oC -180 oC for about 2 hours
(Holladay et al., 2007). The alkaline cooking liquor (‘’white liquor’’) is mixed with the
wood chips in a reaction vessel. After the wood chips have been cooked, the contents of
the digester are discharged under pressure into a blow tank (EPA, 2010). In this process,
pulp is separated from ‘’black liquor’’ (the spent Kraft cooking liquor) (Vishtal and
Kraslawski, 2011). The black liquor is concentrated, typically, by direct contact
evaporation with carbon dioxide, captured from the boiler’s flue stack or with mineral
acids. While lowering the pH, a substantial portion of lignin is precipitated and may be
recovered by filtering and washing (Holladay et al., 2007). Obtained Kraft lignin (Figure
1.5) is structurally highly modified (as approximately 70 % – 75 % of the hydroxyl groups
become sulfonated) and soluble in alkali as well as in highly polar organic solvents (Lange
et al., 2013). It is worth noting, that it is also relatively free from sulphur contamination
(Pandey and Kim, 2011). Kraft lignin constitutes about 85 % of total current world lignin
production. The major part of lignin used to be and still tends to be used in applications
such as production of steam and energy however, in modern Kraft pulp mill incineration
of black liquor produces a surplus of energy. That allows for extraction of part of the
dissolved lignin which can be used for future conversion to high value products (Vishtal
and Kraslawski, 2011).
35
Figure 4.5: Structural characteristics of Kraft pine lignin (Jongerius, 2013).
Lignosulphonate
Sulphite pulping process, can be conducted between pH 2-12, using an acidic mixture of
sulphurous acid, bisulphite ions and calcium, sodium, ammonium or magnesium as the
counter ion (Holladay et al., 2007). In this process, sulphonate groups are introduced in
the lignin structure at the α-position of the propyl side chain and so-called
lignosulphonates are created (Figure 1. 6). Due to the sulphonate groups, most of the
lignin fractions are water-soluble, therefore they differ from other types of lignin
(Gosselink, 2011). Moreover, as a result of incorporation of sulphonate groups in their
structure, they have a higher average molecular weight and monomer weight than Kraft
lignin (Jongerius, 2013). Sulphite pulping does not selectively remove lignin and
carbohydrates appear to be chemically attached to the lignosulphonate fractions. In
some cases, purified lignin fractions can be obtained by the removal of carbohydrate
impurities, using such processes as fermentation, chemical removal, ultrafiltration or
selective precipitation (Gosselink, 2011). Sulphite pulps are less coloured than Kraft pulps
and can be bleached much more easily, but are not as strong. The efficiency and
effectiveness of the sulphite process also depends on the type of wood and the absence
of bark. Mostly, for these reasons, the use of sulphite pulping has declined in comparison
to Kraft pulping over time (EPA, 2010).
36
Figure 4.6: Structural characteristics of lignosulphonate (Jongerius, 2013).
Organosolv lignin
Organosolv pulping is a term, used for the separation of wood components, through
treatment with organic solvents (Lange et al., 2013). Organosolv lignin can be separated
from the pulping solvents by solvent removal and recovery or a combination of
precipitation with water accompanied by distillation to recover solvent (Holladay et al.,
2007). Most organosolv lignin is insoluble in acidic aqueous solutions, but can be
dissolved in many polar organic solvents. Molecular weights are usually less than 1000
and polydispersity may range from about 2.4 to 6.4 (Lange et al., 2013). Organosolv
processes offer lots of possibilities as a source of biorefinery lignin. They are considered
more environmentally friendly than Kraft and sulphite pulping, as they do not require
sulphites (Jongerius, 2013). Higher purity and a lower molecular weight makes
organosolv lignin attractive as a source of low molecular weight phenols or aromatics
(Holladay et al., 2007). A lot of the organosolv pulping processes such as FormicoFib,
Alcell, Acetosolv, Organocell or ASAM have been commercially registered (Vishtal and
Kraslawski, 2011).
The best known variant is the Alcell process (Lora and Glasser, 2002). In this process,
ethanol or the mixture ethanol-water is used as a solvent and due to mild conditions,
37
obtained lignin is pure and unaltered (Pandey and Kim, 2011). The Alcell technology was
demonstrated to successfully produce pulp and lignin already in 1990s (Lora and Glasser,
2002). However, its further development was suspended in 1997 because of financial
difficulties of company supporting this endeavor. Fortunately, the technology has been
acquired by Lignol, which is now being commercialized as a biorefinery technology (Xie
and Gathergood, 2013). Lignol produces ethanol and other biochemicals from hardwood
and softwood species representative of Canadian forests (Menon and Rao, 2012).
Another promising example is the technology developed by the French company CIMV
S.A. This technology uses solvent formed from a mixture of formic and acetic acid
(BIOCORE, 2014). The process is operated at approximately 100 oC and atmospheric
pressure (Leponiemi, 2011). The principal advantage of Organosolv developed by CIMV
is that it forms separate streams of cellulose, hemicellulose and lignin, allowing
valorisation of all components of biomass (Jongerius, 2013). Also, the technology
tolerates a wide variety of biomass feedstocks, including: cereal by-products, forestry
residues and short rotation woody crops. It has been proposed, that CIMV technology
will lead to production of biomass intermediates that can be transformed into useful
bio-products on a commercial scale (BIOCORE, 2014).
Soda pulping
Soda-based cooking methods are usually used for processing annual crops such as straw,
bagasse or flax (Vishtal and Kraslawski, 2011). These methods require heating biomass
in a pressurised reactor to 140 oC -170 oC, mostly in the presence of sodium hydroxide.
Lignin recovered through extraction with sodium hydroxide is normally called ‘soda
lignin’ (Doherty et al., 2011). Soda lignin is sulphur-free and thereby its chemical
composition is closer to natural lignin in comparison with Kraft lignin and
lignosulphonates (Vishtal and Kraslawski, 2011). However, spent soda pulping liquors
very often contain silica, which may co-precipitate with lignin and therefore decrease its
quality (Lora and Glasser, 2002). To solve this problem, the Lignin Precipitation System
process has been developed by Granit S.A, Switzerland and first operated at Papeteries
du Leman, France in 2002 (Pye, 2005). Its main goal was to obtain soda lignin with
relatively low ash and silica content (Lora and Glasser, 2002). In this process, the liquor
is filtered, acidified, aged and filtered again to separate lignin (Pye, 2005). LPS lignin
38
recently became commercially available in powder and solution form (Lora and Glasser,
2002).
1.3.3 LIGNIN FROM BIOMASS CONVERSION PROCESSES FOR BIOREFINING
The biomass-to-ethanol process is regarded one of the potential solutions for the
worldwide need for environmentally sustainable energy resources (Li et al., 2009).
Various biomass conversion technologies have advanced to demonstration and pilot
scale and some lignin products originating from such operations are commercially
available (Lora and Glasser, 2002). There are a few types of biomass conversion
technologies. The biomass conversion technologies used typically are based on biomass
pre-treatment (Wettstein et al., 2012).
Pre-treatment is an important, initial step in a biorefinery operation (Zakzeski et al.,

2010). The main aim of lignocellulosic biomass pre-treatment is to decrease the
crystallinity of cellulose, increase biomass surface area, remove hemicellulose and break
down the lignin barrier (Figure 1.7). Pre-treatment makes cellulose more accessible to
hydrolytic enzymes, therefore rapid conversion of carbohydrate polymers into
fermentable sugars is possible with greater yield (Verma et al., 2011).
A wide range of pre-treatments have been reported and can be divided into several
categories (Goel and Wati, 2013). The most popular pre-treatments include physical,
physico-chemical, chemical and biological pre-treatments (Kumar et al., 2009). Physical
pre-treatment is usually applied to increase the accessible surface area and pore size of
lignocelluloses and to decrease the crystallinity and degree of polymerisation of the
cellulose, which is present in lignocellulose (Behera et al., 2014). Different types of
physical processes include milling, chipping, grinding or irradiation (Kumar et al., 2009).
Pre-treatments that combine both the chemical and physical processes are mostly
important for dissolving hemicellulose and alteration of lignin structure. This category
includes such processes as steam explosion, ammonia fibre explosion, CO2 explosion,
ionic liquid extraction, wet oxidation or liquid hot water (Behera et al., 2014).
In turn, chemical pre-treatments have recently become one of the most promising
methods to improve the biodegradability of cellulose by removing lignin/hemicelluloses
39
as well as decreasing the degree of polymerisation and crystallinity of the cellulosic
component in lignocellulose. Examples of chemical pre-treatment include ozonolysis,
dilute acid hydrolysis or alkaline hydrolysis (Kumar et al., 2009).
Biological pre-treatment is mostly associated with the action of microorganisms that are
capable of producing enzymes, which can degrade lignin, hemicellulose as well as
polyphenols present in biomass. Examples of microorganisms which have been already
reported by researchers to degrade lignin, hemicellulose and to a small extent, cellulose
include mainly brown-, white- and soft-rot fungi, actinomycetes and some types of
bacteria (Behera et al., 2014).
Most pre-treatment techniques in the context of lignin have already been well
investigated and can be divided into a number of pathways (Kumar et al., 2009). Pre-
treatment methods such as steam explosion, ammonia fibre explosion or dilute acid
remove a small amount of lignin from the lignocellulosic substrates. Lignin can be
recovered after enzymatic hydrolysis of polysaccharides. The soluble sugars can be used
for biofuel production and the recovered lignin can be converted to bio-products. In turn,
chemical pre-treatments, for example based on alkaline hydrolysis (lime pre-treatment),
aim at extracting/removing lignin prior to enzymatic saccharification. The bulk of the
biomass lignin is dissolved in the pre-treatment liquor and can be easily separated for
bio-product development. It is worth mentioning, that apart from biomass conversions
based on different pre-treatment methods, thermochemical conversion pathways,
mainly referring to biomass pyrolysis processes are also very popular. After biomass
pyrolysis, lignin (pyrolytic lignin) can be precipitated from pyrolysis oil by phase
separation (Zhang et al., 2011). Examples of lignin fractions, from different biorefinery
processes are described below.
40
Figure 4.7: Effects of pre-treatment on lignocellulosic biomass (Harmsen and Huijgen, 2010).
Steam explosion lignin
Steam explosion is a valuable and important technology. It helps to open up the biomass
fibres and improve the recovery of sugars and other useful compounds from biomass
(Stelte, 2013). In this process, biomass is treated with steam (180 oC -230 oC) under high
pressure (14-35 bar) with a short contact period (1-20 minutess), followed by a rapid
pressure release (Lange et al., 2013). Pre-treated material is separated into 2 fractions:
solid fraction (cellulose and lignin) and liquid fraction (hemicellulose-derived sugars, and
lignin degradation products, acetic acid and other components) (Garcia-Aparicio et al.,
2006). Lignin, can be recovered after enzymatic hydrolysis of polysaccharides (Figure 1.8)
(Zhang et al., 2011). It is also possible to extract by alkali washing or with organic solvents
(Lange et al., 2013). With hardwood lignin, the recovery yield is about 90 % of total lignin.
Softwoods are more recalcitrant. However by treating with alkaline peroxide, these
materials can also be a source of lignin (Holladay et al., 2007).
Figure 4.8: Steam exploded lignin obtained from steam explosion after enzymatic hydrolysis (Zhang et
al., 2011).
41
Dilute acid lignin
Dilute acid pre-treatment typically employs 0.4 % - 2 % H2SO4 at temperatures of 160 oC

-220 oC to remove hemicelluloses and enhance cellulose digestion of cellulose
(Sannigrahi et al., 2010). During these process, the lignin-containing stream is also
obtained as a by-product (Holladay et al., 2007). Potentially, the dilute acid process
provides quite effective separation of lignin from other biomass components (Jongerius,
2013). However, the yield of lignin is pretty low and ranges from 50 % - 70 %. Also, it is
worth mentioning that isolated lignin fractions contain sugar contaminants, which can
be detrimental if used for some applications (NNFCC, 2009).
Pyrolytic lignin
Pyrolysis is the fundamental chemical reaction process that is a precursor of both: the
gasification and combustion of solid fuels. But simply, pyrolysis is defined as the chemical
changes occurring when heat is applied to a material in the absence of oxygen (Fatih
Demirbas, 2009). Pyrolysis can be used to produce a lignin stream for potential use in a
biorefinery. When applied under controlled fast pyrolysis conditions, (temperature of
reaction about 400 oC -450 oC, rapid heating and cooling rates with a short v residence
time) it is possible to generate a bio-oil from whole biomass in yields of up to 75 weight
% on a dry feed basis. The by-products from pyrolysis are char and gas and they can be
used within the process to provide the heat requirements. Also a water fraction can be
generated. It is possible to isolate ‘‘pyrolytic lignin‘‘(a dry powder) from bio-oil, by adding
pyrolysis oil to ice-cooled water while stirring (Figure 1.9). After washing and drying
under vacuum, a light to dark brown powder is obtained (Holladay et al., 2007). It has
been proposed, that removal of pyrolytic lignin from bio-oil can facilitate the
downstream upgrading process and provide a new stream for the development of by-
products (Zhang et al., 2011).
Figure 4.9: Pyrolytic lignin obtained from thermochemical biomass conversion (Zhang et al., 2011).
42
Undoubtedly, lignin obtained using different conversion technologies will have different
characteristics and this will also affect potential products. However, as biomass-to-
biofuel conversion technology is still in the development stage, looking into the
synergetic production of valuable co-products of lignin along with biofuel production can
greatly improve the economic viability of the biomass conversion process (Zhang et al.,
2011).
1.4 APPLICATIONS OF LIGNIN IN INDUSTRY
Current and potential applications of lignin, have been already reviewed in several
reports. However, the most common list of products, which can be derived from lignin
has been developed by Holladay et al., (2007) who separated them into three categories:
(1) power, fuel and syngas products, (2) macromolecules and (3) low-molecular weight
aromatic or phenolic compounds (Figure 1.10) (Zhang et al., 2013).
The first category (near-term opportunities) mainly represents use of lignin as a carbon
source for energy production or focuses on its conversion into energy carriers such as
syngas (Gosselink, 2011). The second category (medium-term opportunities) seeks to
take advantage of the macromolecular structural properties in high-molecular weight
applications (Holladay et al., 2007). The main aim of third category (long-term
opportunities) is to use appropriate technologies to cleave the lignin structure into
monomers, without sacrificing the aromatic rings for the production of polymer building
blocks or aromatic monomers such as benzene, toluene, xylen (BTX), phenol or vanillin
(Gosselink, 2011).
The concept of near-, medium- and long-term shows probabilities for application of
lignin over the short- to long-term. ‘’Near-time’’ is defined as current applications and
those that are probable within three to ten years. Some technology development will be
needed, but much can be borrowed from processes which are already well-established,
for example pyrolysis. ‘’Medium-term’’ refers to the period 5 to 20 years ahead and
requires more significant development of technology as well as further research to
increase fundamental knowledge of the area. In turn, ‘’long-term’’ refers to beyond
about 20 years and requires new technology development and novel research in the
area (Holladay et al., 2007). Each of the categories is discussed below and current
43
technologies, products derived from lignin and the major challenges ahead are
considered.
Figure 4.10: Valuable products obtained from lignin (adapted from Yuan et al., 2013 ).
1.4.1 NEAR –TERM OPPORTUNITIES: POWER, FUEL AND SYNGAS PRODUCTS
The main aim of near-term opportunities for lignocellulosic biorefineries, is to convert

lignin to power, liquid fuel and syngas products (Table 1.1) (Holladay et al., 2007). This
can be achieved by several methods and they include: combustion, gasification, pyrolysis
and hydroliquefaction (Zhang et al., 2011).
1.4.1.1 Combustion
Combustion, commonly known as burning is a chemical reaction between fuel and
oxygen .The main products of this process are carbon dioxide, water and heat (Naik et
al., 2010). Lignin combustion, is already well-practiced in paper mills and can be also
applied in lignocellulosic biorefineries (Holladay et al., 2007).
1.4.1.2 Gasification
The gasification process involves a reaction of biomass with air, oxygen or steam to
produce a gaseous mixture of carbon monoxide (CO), carbon dioxide (CO2), hydrogen
(H2), methane (CH4) and N2, known as producer gas, synthesis gas or syngas. The name
depends on the proportion of component gases (Naik et al., 2010). Syngas can be used
in different ways. One well-established technology produces methanol/dimethyl ether
(DME). Obtained products can be used directly or be converted to gasolines via methanol
to gasoline (MTG) process or to olefins via the methanol to olefin (MTO) process. Fischer-
Tropsh technology (FT) produces green diesel from lignin-derived syngas. Conversion of
syngas to mixed alcohol has not been commercialised yet. However, it would allow
44
production of ethanol and other alcohols or alcohol derived chemicals (Holladay et al.,
2007).
1.4.1.3 Pyrolysis
Pyrolysis is a thermal degradation of biomass by heat in the absence of oxygen, which
results in the production of charcoal, bio-oil and gaseous fuel products. There are three
different types of pyrolysis, depending on the operating conditions: conventional
pyrolysis, fast pyrolysis and flash pyrolysis (Naik et al., 2010). Fast pyrolysis is a process
that can convert dry biomass to liquid product known as pyrolysis oil or bio-oil. Pyrolysis
oil (including lignin) can be suitable for integration into an alcohols or alcohol chemicals
petroleum refinery (Holladay et al., 2007).
1.4.1.4 Hydroliquefication
Lignin can also be converted into fuels through hydroliquefication (Beauchet et al.,
2012). This method has been developed by NREL and is a multi-step process to convert
lignin into a branched aromatic hydrocarbon product, which can be used as a blending
component for reformulated gasoline. The first step of this process, called base-
catalysed depolymerisation, breaks the lignin polymer into phenolic intermediates,
which can be hydroprocessed into a final product (Holladay et al., 2007). These steps
include hydrodeoxygenation and hydrocracking (Pandey and Kim, 2011). The final
gasoline-blending component consists of a mixture of naphthenic and aromatic
hydrocarbons (Holladay et al., 2007).
45
Table 4.1: Overview of technology, product examples and challenges for conversion of lignin to power-
fuel-syngas products (adapted from Holladay et al., 2007).
1.4.2 MEDIUM-TERM OPPORTUNITIES: MACROMOLECULES
The main aim of medium-term opportunities is to maintain the macromolecule structure

of lignin in high-molecular weight applications. Lignin can mostly be used in these
applications directly (Table 1.2). However, sometimes chemical modification is required
(Holladay et al., 2007). Examples of applications from lignin as a macromolecule include
carbon fibres, polymer modifiers and resins/adhesives and binders (NNFCC, 2009).
1.4.2.1 Carbon fibres

Carbon fibres are used for light and strong materials in e.g. aerospace, defence,
recreation or the general industrial market (LigniMATCH, 2010). Sales of carbon fibres
are expected to increase from $1.6 billion to $4.5 billion in 2020. In terms of applications,
global sales of carbon fibre reinforced plastics were estimated at $16.1 billion in 2001
and are forecasted to reach $28.2 billion in 2015 and $48.7 billion by 2020 (Smolarski,
2012). One thing is sure; the high cost of this material significantly reduces production
of carbon fibres. Therefore, undoubtedly any other alternative cheaper raw material is
desirable (LigniMATCH, 2010). Lignin represents a potential, low-cost source of carbon,
suitable for displacing synthetic polymers such as polyacrylonitrile (Holladay et al., 2007).
Potentially, lignin-derived carbon fibres would be attractive to the automotive industry
46
at a price in the order of $6.5-11/kg (LigniMATCH, 2010). It has been proposed, that
“diverting 10 % of the lignin potentially available in the US could produce enough carbon
fibre to replace half of the steel in domestic passenger vehicles” (Smolarski, 2012).
Developing methods for the effective utilisation of lignin in carbon fibre applications is
being currently studied in the USA by Oak Ridge National Laboratory and in Sweden
(NNFCC, 2009).
1.4.2.2 Polymer modifers

Lignin can find lots of applications in thermoplastic and thermosetting industries either
as a low cost filler or higher-value additive (NNFCC, 2009). Currently use of lignin focuses
on the fillers. However, future research should concentrate on the latter by creating
technology that improves polymer alloying, mutual solubility or crosslinking. Eventually,
the modified lignin can be used for example, in high-strength engineering plastics, heat
resistant polymers or antibacterial surfaces (Holladay et al., 2007).
1.4.2.3 Resins/Adhesives and binders

Lignin is a natural glue with an aromatic and highly-crossed linked structure, similar to
the network structure of phenol-formaldehyde resins (NNFCC, 2009). However,
formaldehyde is currently considered a carcinogen (Holladay et al., 2007). The toxicity
and cost of formaldehyde and stricter control over its use, have led the search for safe,
emissions free alternatives. Lignin modification to provide enhanced properties has been
already successful demonstrated in the production of epoxy, phenolic resins and
adhesives (with similar or enhanced properties compared to conventional methods). The
problem is that widespread use of lignin as a resin/adhesive is limited by its chemical
heterogeneity or the presence of contaminants. Therefore, applications for lignin in this
area are still a technical challenge (NNFCC, 2009).
47
Table 4.2: Overview of challenges for the use of lignin as a macromolecule (adapted from Holladay et al.,
2007).
1.4.3 LONG-TERM OPPORTUNITIES: LOW MOLECULAR WEIGHT AROMATIC OR PHENOLIC COMPOUNDS
The market demands for aromatic and phenolic compounds are enormous (Holladay et
al., 2007). Benzene, toluene and xylene (BTX) are the sources of about 60 % of all
aromatics in volume. They represent a 100-billion dollar market and are produced in the
following amounts: benzene-40.2 million tonnes per year, toluene-19.8 million tonnes
per year and xylene-42.5 million tonnes per year. The average price for BTX is about
$1200 per tonne. Moreover, the BTX market is forecasted to grow significantly from
2010 to 2020 (Smolarski, 2012). Phenol demand has grown at a rate of 5 % since 2001
(NNFCC, 2009). The current phenol market price value is about $1500 per tonne. Two
thirds of phenol production involves conversion to plastics or related materials (for
example phenol formaldehyde resins, polyurethane foams or polyurethanes for
automobiles). With regard to vanillin, the global demand for this product was estimated
at around 16,000 tonnes per year in 2010. Annual demand for vanillin is growing at a
stable annual growth rate of 2 % in Europe and USA. (Smolarski, 2012).
48
It has been proposed, that depolymerizing lignin to low-molecular weight aromatic and
phenolic compounds can offer the greatest opportunity to truly expand the spectrum of
lignin applications and dispel the lignin myth (Gosselink, 2011). Currently, phenol is
directly affected by the price of oil. However, the lignin price is quite stable and is likely
to decrease as yields improve and technology becomes available (Smolarski, 2012).
Phenolic compounds derived from depolymerisation of lignin can be used for replacing
phenol in phenolic resins. Moreover, many types of monomeric and oligomeric subunits
of lignin are recognized for their antimicrobial or anticorrosive properties (Gosselink,
2011). It is worth mentioning, that phenols are very flexible in terms of source, and the
phenolic substances from lignin, do not differ in potential application from the fossil-
derived molecules (LigniMATCH, 2010).
A number of different conversion methods have been proposed to depolymerize lignin

to low-molecular weight compounds. They can be divided into three groups: biological,
chemical and thermal methods. Biological depolymerisation can be carried out using
fungi (white rot fungi). Fungal decay of wood results in breaking of bonds in lignin by
enzymes assisted by other environmental influences or bacterial consortia. Chemical
depolymerisation includes base-catalysed depolymerisation, acid-catalysed
depolymerisation or oxidative depolymerisation. When considering thermal methods of
lignin depolymerisation, it is worth mentioning methods such as: pyrolysis,
hydrodeoxygenation, solvolysis or supercritical depolymerisation (Gosselink, 2011).
However, the challenge of lignin depolymerisation still is an issue. Lack of effective and
cost competitive methods currently prevents realization of lignin’s full potential.
Undoubtedly, challenges in this research area are ongoing (Zhang et al., 2011). An
overview of product examples for aromatics, their market nichesand challenges are
shown in Table 1.3 and 1.4.
49
Table 4.3: Overview of product examples and challenges for aromatics, including benzene, toluene and
xylene (BTX), phenols, monomeric lignin molecules and other low-molecular weight products (adapted
from Holladay et al., 2007).
50
Table 4.4: Phenol and derivatives; production, market niche and application (Gosselink, 2011).
1.5 VALORISATION OF LIGNIN

1.5.1 CURRENT MARKETS
There are only a few existing markets for lignin. Most of lignin sold today is
lignosulphonate derived from sulphite processing (NNFCC, 2009). The world leading
lignosulphonate producer is Borregaard (Norway) (Holladay et al., 2007). Its annual
production is more than 500,000 tons of lignosulphonate solids (Vishtal and Kraslawski,
2011). In turn, MeadVestaco Corporation in U.S is the main supplier of Kraft lignin. The
chart below shows the uses of Kraft lignin and lignosulphonate with the main application
being as a fuel (Figure 1.11).
Only one million tonnes of lignosulphonates (from 70 million tonnes/year) and less than
100,000 tons of Kraft lignin (from 4milion tonnes/year) are isolated annually and sold for
non-fuel applications. In the case of Kraft lignin, the main non-fuel applications are as a
rubber reinforcer or activated carbon. In the case of lignosulphonates, non-fuel
applications include animal feed pellets, or concrete admixtures (Tuomi et al., 2010).)
51
Figure 4.11: Main uses of Kraft lignin and lignosulphonate (Tuomi et al., 2010).
1.5.2 FUTURE MARKETS
Production of Kraft pulp is expected to increase by 1.4 % each year in the period from
2005 to 2020. It would result in a potential 56 million tonnes of Kraft lignin available
globally by 2025 (NNFCC, 2009). Apart from the potential growth of Kraft lignin, there is
also a major opportunity for organosolv lignin; its high purity and certain properties
compared to lignosulphonate might enable this product to enter new market sectors,
where other lignin fractions are not applicable due to smell, colour, heterogeneity or
purity. Organosolv lignin has prospective new market areas in the food, cosmetic or
pharmaceutical industries (Tuomi et al., 2010). It has already been proven, that the type
of process used to produce lignin can potentially dictate the value of the products that
can be derived from it. The chart below (Figure 1.12) shows a strong correlation between
the price of each type of lignin and the added value of the final products. Undoubtedly,
Kraft lignin and organosolv lignin appear to be the most suitable/promising candidates
for the expansion of the lignin sector (Smolarski, 2012).
There is a wide range of different potential competing markets for isolated lignin,
including production of biofuels and other bio-derived chemicals or materials. The
relative strength of markets now and in the near future will definitely affect the price
and availability of lignin. Therefore full valorisation is needed to determine the best
products to make lignin economically valuable (NNFCC, 2009).
Valorisation of lignin plays a very important role in the further development of cost
effective biorefinery processes for biofuels and the production of bio-based materials
52
and chemicals from lignocellulosic biomass (Gosselink, 2011). Unfortunately, only 2 % of
all industrial lignin are valorised per year (Schorr et al., 2014). The lignin market today
represents about $300 million. However, the total lignin availability exceeds 300 billion
tonnes and increases annually by around 20 billion tonnes. Economic analysis shows,
that use of lignin for energy applications alone is not economically viable. Therefore
lignin-derived chemicals and other building blocks need to be upgraded to use in final
products. Potential applications include different polymeric materials and resins
(Subsidie and Termijn, 2011).
Nowadays, the second largest outlet for phenol is phenolic resins, which can account for
about 27 % of phenol use. This enormous demand for phenolic resins, also represents
the potential growing market for lignin (NNFCC, 2009). Moreover, there are many
favourable factors in the case of lignin in contrast with phenols derived from the
petrochemical industry. This includes its abundance and availability, antioxidant and
antimicrobial properties, biodegradable nature, reinforcing capability, as well as
environmental sustainability e.g. carbon neutral, making it an ideal candidate for the
development of novel polymer composite materials (Thakur et al., 2014).
Undoubtedly, lignin can also be used in plastic materials. Currently, 230,000 tonnes of
petroleum-derived plastics are produced per year (Schorr et al., 2014). Lignin-based
materials are already used in combination with petroleum–based materials to increase
biodegradability of plastics. Unfortunately this is currently on a small scale, for example
in the electronic industry or in concept cars, as the commercial breakthrough for using
lignin in plastic has not been fully realised (LigniMATCH, 2010). However, it has been
estimated that production of new polyolefin biocomposites containing lignin could
contribute both to creation of new value added products and to a reduction in the use
of petrochemicals, non-renewable raw materials. These products could help to improve
the economic situation in the pulp and paper industry, which is in the process of
integrating the biorefinery concept. This could also help to develop technology in the
eco-materials field and contribute to a decrease in greenhouse gases by partial
replacement of petrochemicals with natural products (Schorr et al., 2014).
53
Figure 4.12: Lignin value vs lignin-based product value by lignin type (Gosselink, 2011).
1.6 BIO-BASED PRODUCTS- OPTIMISATION OF LIGNIN PRESCURSORS THROUGH PHYSICO-

CHEMICAL AND BIOLOGICAL MODIFICATION
Several studies have previously been carried out on the conversion of lignin to value-
added products. Pouteau et al., 2003., Falkehag, 1975 and De Chirico et al., 2003
investigated the incorporation of lignin into industrial thermoplastics to reduce the costs
of polymer production. In turn, the outcomes of the reserach performed by Yu et al.,
2006., Bismarck et al., 2006 and Pouteau et al., 2004 suggested that lignin can be directly
used with elastomers or polyolefins as an antioxidant, ultraviolet light stabiliser and that
it also has potential as a flame retardant. However, it has been demonstrated, that use
of llignin in its extracted form is not always very effective (Laurichesse and Avérous,
2013). It is well recognised that lignin is highly resistant towards chemical and
biochemical degradation processes and it is therefor desirable to develop new
technologies that can bring about the successful modification of this macromolecule
(Tian et al., 2010).
54
1.6.1 LIGNIN MODIFICATION AND DEGRADATION BY PHYSICO-CHEMICAL METHODS
Currently, physico-chemical modification of lignin appears to be one of the potential

routes to exploit this renewable product as a starting material for synthesis of
biomaterials. This can be classified into three categories: (1) lignin depolymerization to
aromatics or fragmentation to lower molecular weight compounds, (2) chemical
modification to create new active sites, (3) functionalization of hydroxyl groups
(Laurichesse and Avérous, 2013). Schematics showing different routes for physico-
chemical modification of lignin are presented below (Figures 1.13, 1.14 and 1.15).
Examples of the most common and emerging technologies for improving the properties
of ignin are described in detail. Commented [AW[3]: DMSO?
Figure 4.13: Examples of the main processes for lignin fragmentation (adapted from Laurichesse and
Avérous 2013).
55
Figure 4.14: Examples of the main routes for the synthesis of new chemical active sites (adapted from
Laurichesse and Avérous, 2013, Cateto et al., 2008).
Figure 4.15: Examples of the chemical reactions for the functionalization of lignin hydroxyl groups
(adapted from Laurichesse and Avérous 2013).
Phenolation
In this process, additional phenol groups can be attached to benzylic carbons of lignin.
The phenolation reaction is carried out in acidic medium and yields a lignin with
additional ortho and para reactive sides on the added phenol groups. Side chain
reactions cause lignin fragmentation, resulting in a decrease in the molecular weight that
promotes the incorporation of the phenolated lignin into polymeric strutures such as the
resin network (Duval and Lawoko, 2014). So far it has been observed, that lignin-based
PF-resins with 33 % phenolated bagasse lignin produced laminates with better physical
properties than unmodified lignin. Phenolation of rice hull acid-insoluble lignin also
56
exhibited improved properties compared to unmodified lignin-based PF-resins with
regard to morphology and thermal stability (Ghaffar and Fan, 2014).
Ultrafiltration (lignin purification)
Studies show, that technical lignin fractions purified by ultrafiltration are promising
materials for lignin-based PF-resins, and this relatively low cost process can make them
very feasible and economically attractive. There are two main advantages of
ultrafiltration: adjustment of the pH or temperature is not needed, and the
concentration of the liquor to be treated is not crucial (Jönsson and Wallberg, 2009).
Chemical catalysis
Catalysis has also recently been considered as an important technology in biomass and
lignin conversion. Catalysts applied in lignin depolymerization can promote high
conversion rates and suppress char formation or condensation while minimizing the
harshness of reaction conditions. Currently, zeolites and silica-alumina catalysts appear
to be among the most promising in the catalysis process. They disrupt the lignin by
cracking and have been applied for upgrading of lignin pyrolysis oil. Amorphous silica-
alumina catalysts promote the production of aliphatic hydrocarbons, and zeolite H-ZSM
produce more aromatic than aliphatic hydrocarbons (Pandey and Kim, 2011).
Lignin solubility in organic solvents
The limited solubility of lignin in organic solvents is one of the key factors restricting their
use as an adhesive. It has already been shown, that the solubility of lignin (for example
Kraft lignin) can be improved by partial depolymerization through base-catalyzed
depolymerization in supercritical methanol. The partially depolymerized lignin can then
be converted to lignin-based polycarboxylic acid (LPCA) in the presence of succinic
anhydride. Afterwards, LPCA has the potential to be used for a commercial epoxy (Ten
and Vermerris, 2015).
57
Graft copolymerisation
Another emerging technology in the area of chemical modification of lignin is graft
copolymerization (Thakur et al., 2014).The main aim of this process is to attach polymer
chains to the lignin hydroxyl groups, thus resulting in a star-like branched copolymer,
with the lignin core. Two routes can be used for the synthesis of graft copolymers:
(Figure 1.16) (Duval and Lawoko, 2014):
a) “Grafting from: there are two types of polymerization reactions, which are performed
in order to produce lignin-graft copolymers by the ‘grafting from’ route. The first involves
ring opening polymerization (ROP) of different monomers (such as propylene oxide, ε-
caprolactone, lactide) initiated by lignin hydroxyl groups. The second involves the radical
polymerization of vinylic monomers, such as vinyl acetate, onto the lignin
macromolecule. isocyanate groups at one end.
b) “Grafting onto”- in order to react with lignin OH functions, the polymer chain is first
synthesised, and functionalized at one end with a chemical function able to react with
lignin OH groups (for example with diisocyanate, to obtain polymer chains bearing free
isocyanate groups at one end). The polymer chains are then grafted to the lignin core.
Attempts for a copolymerization process have been conducted by researchers from

Stanford University. In their study, lignin was modified using a graft copolymerization
reaction, catalyzed by triazabicyclodecene (TBD). It has been observed that grafted lignin
is soluble in organic solvents and the data demonstrate that graft copolymerization may
play a key role in altering the surface characteristics of lignin, making it compatible with
different applications (Thakur et al., 2014).
58
Figure 4.16: Schematic showing synthesis of lignin graft copolymers: a) grafting from, b) grafting onto
(Duval and Lawoko, 2014).
1.6.2 LIGNIN MODIFICATION/DEGRADATION BY MICROORGANISMS AND THEIR ENZYMES
Enzymatic methods are amongst the most promising areas for the development of new
processes. These bioconversion processes involve use of different microorganisms or
their enzymes for the modification and degradation of lignin polymers (Buranov and
Mazza, 2008). They can contribute to a more efficient and environmentally sound use of
renewable feedstocks for sustainable production of materials and also chemicals,
biofuels or energy (Ruiz-Dueñas and Martínez, 2009). Compared to other physical or
chemical methods they show a high selectivity for lignin over cellulose (Nazarpour et al.,
2013). Different microorganisms, examples of enzymes and their functionalities are
described below and are shown in Table 1.5.
1.6.2.1 Lignin degradation by fungi

Lignin can be effectively broken down by a group of fungi known as white-rot fungi,
brown-rot fungi and soft-rot fungi (Swe, 2011). Wood-rotting fungi are the only
eukaryotic organisms that produce laccase and peroxidase enzymes and therefore play
a crucial role in the degradation of plant material (Knežević et al., 2013).
1.6.2.1.1 White-rot fungi

Wood rotting white-rot fungi are the most efficient lignin degraders in nature and have
a capacity to remove lignin that makes them ideally suited for industrial applications (Qi-
59
He et al., 2011) and are comprised of mostly Basidiomycetes and a few Ascomycetes
(Crawford, 1980). Analysis of white-rot degraded wood shows, that the reactions with
lignin: involve demethylation (or demethoxylation), are oxidative, include side-chain
oxidation at Cα and involve propyl side-chain cleavage between Cα and Cβ (Geib et al.,
2008). The key enzymes that function in lignin degradation are phenol oxidases. Of
these, laccase (Knežević et al., 2013), lignin peroxidases (LiP), manganese peroxidases
(MnP), versatile Peroxidase (VP) are quite well-known (Ruiz-Dueñas and Martínez, 2009.,
Janusz et al., 2013). Ligninolytic enzymes of white-rot fungi have mostly been reported
to be extracellular but there is evidence in the literature of the occurrence of intracellular
laccases in white–rot fungi (Kunamneni et al., 2011) and also other intracellular enzymes
involved in the bioconversion of lignin derivatives both in fungi and bacteria (Gellesterdt,
2009; Christopher et al., 2014; Pollegioni et al., 2015).
Classes of ligninolytic enzymes:
Lignin peroxidase (LiP): Lignin peroxidase, mostly produced by Phanerochaete

chrysposporium is a heme-containing enzyme, capable of cleaving C-C as well as ether
bonds, using hydrogen peroxide as the oxidant (Franssen et al., 2013). Its main role is to
degrade non-phenolic units of lignin (Martínez et al., 2005).
Manganase Peroxidase (MnP): Manganese peroxidase (MnP) is a heme-containing

glycoprotein, first identified in P. chrysosporium (Martínez et al., 2005) and is secreted
by 56 known fungi often produced in multiple isoforms. For instance 11 have already
been described in one fungal species, Ceriporiopsis subvermispora, which differ in their
isolectric points. The Mn peroxidases catalyse the oxidation of complexed Mn 2+ to Mn
3+, which can then oxidize a large number of phenolic substrates. Mn3+ is widely accepted
as a diffusible oxidant. It is able to oxidize secondary substrates at a distance away from

the active site of MnP (Bugg et al., 2011a).
Both LiP and MnP enzymes catalyse the oxidation of the recalcitrant non-phenolic lignin
units by H2O2. The two aspects giving them the catalytic properties are: a) a heme
cofactor, conferring high redox potential to the oxo-ferryl complexes, b) existence of
specific binding sites for oxidation of their characteristic substrates, including non-
60
phenolic aromatics in the case of LiP and manganous iron in the case of MnP (Martínez
et al., 2005).
Versatile Peroxidase (VP): The third type of peroxidase is produced mainly by Pleurotus
spp. This enzyme has a catalytic versatility and ability to degrade high-redox potential
aromatic compounds (for example polycyclic aromatic hydrocarbons, phenolic and non-
phenolic aromatic pollutants, pesticides or dyes) which other LiP and MnP cannot oxidize
directly (Ruiz-Dueñas and Martínez, 2009).
Laccase: Laccases catalyse the oxidation of various phenolic substrates via the reduction
of oxygen to water (Lahtinen et al., 2013). The active site of laccase includes four copper
ions. Type-I copper serves as an electron acceptor from substituted phenols or amines.
In turn, type II transfers the electrons to the final acceptor, dioxygen which is reduced
to water (Martínez et al., 2005). Laccases cleave phenolic lignin model compounds and
nonphenolic compounds via the mediator system (Bugg et al., 2011a). A mediator is a
small chemical compound that acts as a sort of ‘electron shuttle’: once it is oxidized by
the enzyme generating a strongly oxidizing intermediate (oxidized mediator), it diffuses
away from the enzymatic pocket and in turn oxidizes any substrate, that due to its size
could not directly enter into the active site (Kunamneni et al., 2011). Laccase can be
produced for example by T.versicolor and Phlebia radiata in addition to peroxidases
(Bugg et al., 2011a).
Other enzymes
In addition to ligninases, other extracellular enzymes which act as accessory enzymes

have been found and they include aryl-alcohol oxidase (AAO) found in Pleurotus eryngii
or glyoxal oxidase (GLOX) in P. chrysosporium. Moreover, aryl-alcohol dehydrogenase
(AAD), quinone reductases (QR), cellobiose dehydrogenase (CDH) (Dashtban et al., 2010)
and dye-decolorizing peroxidase (Camarero et al., 2014) have been also observed.
Among all of the white-rot fungi species, P. chrysosporium is one of the most investigated
basidiomycetes (Bugg et al., 2011a). It plays a key role in the degradation of lignin
polymers to vanillin, vanillic acid, coniferyl aldehyde and other products (Buranov and
Mazza, 2008). It has 16 candidate genes associated with lignin degradation; 10 LiP
enzymes, 5 MnP enzymes and 1 NoP (novel peroxidase) (Bugg et al., 2011a).
61
1.6.2.1.2 Brown-rot fungi
Brown-rot fungi include only 7 % of all Basidiomycetes (Bugg et al., 2011a). Their main
role is degradation of wood polysaccharides, whereas lignin is degraded only slightly.
This separates brown-rot fungi physiologically from the white-rot group (Crawford,
1980). The brown-rotters do not cleave lignin's aromatic rings efficiently and therefore
are unable to achieve significant decomposition resulting in lignin fragments (Mahesh
and Mohini, 2013).
1.6.2.1.3 Soft-rot fungi

A variety of Ascomycetes and Fungi Imperfecti are included among the soft-rot fungi
(Crawford, 1980). Compared to white-rot and brown-rot fungi, the literature is scant
regarding the mechanism of lignocellulose degradation by soft-rot fungi. However, it is
clear, that some of the species degrade lignin, as they erode the secondary cell-wall and
decrease the content of acid-insoluble material (Klason lignin) in angiosperm wood
(Sánchez, 2009). Examples of species previously observed in the process of lignin
degradation include: Graphium, Monodictys, Paecilomyces, Papulospora, Thielavia and
Allescheria (Crawford, 1980). Recent studies, also show the involvement of species such
as Neurospora crassa and Trichoderma sp. producing laccase (Kunamneni et al., 2011;
Gochev and Krastanov, 2007; Hölker et al., 2002) and Chaetomium sp. producing β-
etherase that cleaves β-0-4-aryl-ether bonds, which account for 45 %-60 % of all linkages
in lignin. The use of optimised β-etherase could enable a more specific and effective
pathaway for lignin depolymerisation as well as valorization (Picart et al., 2016).
Typical enzymes:
β-etherase: This enzyme catalyzes the cleavage of β-0-4-aryl-ether bonds, which account
for 45 %-60 % of all linkages in lignin. The use of optimised β-etherase could enable a
more specific and effective pathway for lignin depolymerisation as well as valorization
(Picart et al., 2016).
Other enzymes
Apart from the above mentioned enzymes, novel white rot laccase was also recently
purified from the soil fungus Myrothecium verrucaria (Pollegioni et al., 2015).
62
1.6.2.2 Lignin degradation by bacteria
Bacterial mechanisms of lignin degradation and their ligninolytic enzyme pathways are
an ongoing topic of research (Buranov and Mazza, 2008; Gellesterdt, 2009; Bugg et al.,
2011a; Christopher et al., 2014; Ding et al., 2015; Pollegioni et al., 2015). As the initial
results show, Pseudomonas can cause lignin degradation to guaiacol and vanillic acid
(Figure 1.17) (Buranov and Mazza, 2008). It has been suggested, that bacteria might use
similar types of extracellular lignin-degrading enzymes to modify lignin (Huang et al.,
2013). In the supernatants of ligninolytic bacteria cultured with lignin, both peroxidase
and laccase enzyme activities have been detected (Bugg et al., 2011b). Several
Streptomycetes have previously been reported to break down lignin, of which the best-
studied species is Streptomyces viridosporus, producing some extracellular peroxidases,
which have been shown to catalyze oxidative cleavage of β-aryl ether lignin model
compounds (Bugg et al., 2011a). Examples of species showing intracellular activity of
laccase include Azospirillum lipoferum, Marinomonas mediterranea and Bacillus subtilis
(Christopher et al., 2014). In species such as P. fluorescens, P.putida, Sphingomonas
paucimobilis and Corynebacterium glutamicum the activity of vanilin dehydrogenase,
mostly used in degradation of lignin derived phenylpropanoids, has also been observed
(Ding et al., 2015). Other bacteria capable of lignin degradation include S. paucimobilis
producing β-etherase (Gellesterdt, 2009; (Picart et al., 2016)., vanilin dehydrogenase,
Cα-dehydrogenase (Gellesterdt, 2009) and dioxygenase (Bugg et al., 2011a).
Figure 4.17: Lignin model compound (I) and its degradation to guaiacol (II) and vanillic acid (III) in the
presence of bacteria (Buranov and Mazza, 2008).
63
Typical enzymes:
Dyp-type peroxidase: This type of enzyme in bacteria assumes the role of lignin-
peroxidases (de Gonzalo et al., 2016).
Monooxygenase: The main role of this enzyme is demethylation of vanilic acid and
generation of a new functional OH group (Zhu et al., 2017).
Laccase: In comparision to fungal laccases, bacterial laccases are normally more stable
at high pH and high temperatures. Whereas fungal laccases can be both intra- and extra-
cellular, bacterial laccases are mostly intracellular (Christopher et al., 2014).
Vanilin dehydrogenase: This is a critical enzyme for the degradation of lignin-derived

phenylpropanoids (such as vanillin, vanillate, caffeate, p-coumarate, and cinnamate). It
can also function in the degradation of vanillin, benzaldehyde, p-hydroxybenzaldehyde,
or protocatechualdehyde (Ding et al., 2015).
Cα-Dehydrogenase: This enzyme participates in oxidation of the α-carbon to α-carbonyl

(Gellesterdt, 2009).
Dioxygenase: This enzyme is a non-heme iron-dependent enzyme and catalyses the

oxidative cleavage of lignostilbene, thereby yielding two molecules of vanillin (Bugg et
al., 2011a).
Decarboxylase: This enzyme converts hydroxycinnamic acids into the corresponding 4-

vinyl aromatic compound (Bugg et al., 2011a).
Despite the fact, that certain lignocellulolytic fungi are able to secrete extracellular
enzymes on an industrial scale, bacterial enzyme production can be more cost-efficient.
They grow more rapidly, produce multi-enzyme complexes with increased functionalities
and higher specificities, and can tolerate larger and more diverse environmental stresses
(Woo et al., 2014). Also, even though lignin biodegradation is generally an aerobic
process, some authors also report that anaerobic microorganisms may alter or partially
degrade lignin (Pérez et al., 2002). Among the anaerobic bacterial lignin or phenol
degraders are for example S. paucimobilis or Rhodococcus sp. Furthermore, lignin
degradation in an anaerobic bacterium Enterobacter lignolyticus has been reported
more recently and this discovery has elucidated alternative the natural processes of
64
bacterial lignin decomposition (DeAngelis et al., 2013). It has been demonstrated, that
biotechnology based on lignin-degrading microbes and their enzymes can contribute to
more efficient and environmentally sound use of renewable lignocellulosic feedstocks
for the sustainable production of different materials (Ruiz-Dueñas and Martínez, 2009).
65
Table 4.5: Organisms and enzymes involved in lignin conversion.
Organism Enzyme Effect Cellular location References
Organisms
(Bacteria)
Sphingobium β-etherase Catalyzes the cleavage of extracellular Gellerstedt, (2009),

paucimobilis (β-etherase β-0-4-aryl-ether bonds, Picart et al., (2015)
Lig E, F, G) which account for 45-60
% of all linkages in lignin
Sphingobium 4,5-dioxygenase Catalyses the oxidative Bugg et al., (2011b)

paucimobilis enzyme LigAB cleavage of lignostilbene
to give two
Dioxygenase LigZ molecules of vanillin.
Pseudomonas Vanilin Critical enzyme for the Ding et al., (2015)

fluorescens, dehydrogenase degradation of lignin
Pseudomonas derived
putida, phenylpropanoids (such
Pseudomonas sp, as vanillin, vanillate,
Sphingomonas caffeate, p-coumarate,
paucimobilis, and cinnamate).
Corynebacterium Degradation of
glutamicum benzaldehyde, (p-
hydroxybenzaldehyde,
protocatechualdehyde)
Streptomyces Lignin peroxidase Catalyses oxidative extracellular Bugg et al., (2011b),

viridosporus, (Dye- cleavage of β-aryl ether Dashtban et al., (2010)
Acinetobacter decolourisiing lignin Santos et al., (2014)
calcoaceticus Peroxidase)
Pseudomonas
putida
Pseudonocardia Monooxygenase Demethylation of vanilic Zhu et al., (2017)

acid by monooxygenase
which generates a new
functional hydroxyl group
Sphingomonas Cα- Oxidation of the α- intracellular Gellerstedt, (2009)

paucimobilis Dehydrogenase carbon to a carbonyl
Azospirillum Laccase intracellular Christopher et al.,

lipoferum (2014)
Marinomonas Laccase intracellular Christopher et al.,

mediterranea (2014)
Bacillus subtilis Laccase intracellular Christopher et al.,

(2014)
Streptomyces Laccase Pollegioni et al., (2015)

coelicolor,
Streptomyces
ipomoea,
Streptomyces
lividans
Pseudomonas Laccase Kuddus et al., (2013),

putida Arunkumar et al.,
Pseudomonas (2014)
aeruginosa
66
Sphingomonas Demethylase Bugg et al., (2011a)
paucimobilis
Bacillus sp. Decarboxylase Converts Bugg et al., (2011a)

Enterobacter sp hydroxycinnamic acids
into the corresponding 4-
vinyl aromatic
compounds
Organisms
(White-Rot Fungi)
Phanerochaete Lignin peroxidase Catalyses oxidative extracellular Martinez et al., (2005),

chrysposporium (LiP) cleavage of β-aryl ether Dashtban et al., (2010)
Trametes lignin, degrades non-
versicolor, phenolic lignin unit
Panus sp.
Pycnoporus
sanguineus
Phanerochaete Manganese Generates MN3+ which extracellular Martinez et al., (2005),

chrysposporium peroxidase (MnP) acts as a diffusible Bugg et al., (2011a),
Lenzites betulinus, oxidizer on phenolic or Pollegoni et al., (2015),
Phanerochaete non-phenolic lignin unit Dashtban et al., (2010)
flavido-alba, via lipid peroxidation
Nematoloma reaction
frowardii
Pleurotus , Versatile Combines the catalytic extracellular Martinez et al., (2005),

Bjerkandera, Peroxidase (VP) properties of LiP and MnP Pollegioni et al.,
Pleurotus and plant/microbial (2015), Dashtban et
ostreatus, peroxidases oxidizing al., (2010)
Pleurotus eryngii, phenolic compounds
Pleurotus tigrinus
Trametes Laccase Laccases Martinez et al., (2005),

versicolor cleave phenolic lignin Bugg et al., (2011a),
(Basidiomycete) model compounds and Pollegioni et al., (2015)
nonphenolic
compounds via the
mediator system
Irpex lacteus Dye-decolorizing Degrades non-phenolic Camarero et al., (2014)

peroxidase (DyP) lignin compounds
Organisms
(Brown-Rot and
Soft-Rot Fungi)
Melanocarpus Dye-decolorizing Degrades non-phenolic Martinez et al., (2005)

albomyces peroxidase (DyP) lignin compounds
(Ascomycete)
Chaetomium β-etherase Catalyzes the cleavage of extracellular Picart et al., (2015),

(Ascomycete) β-0-4-aryl-ether bonds,
which account for 45-60
% of all linkages in lignin
Neurospora Laccase extracellular/ Kunamneni et al.,

crassa intracellular (2007)
Laccase
Trichoderma sp. Gochev and Krastanov,
(2007)
Hölker et al., (2002)
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1.7 CONCLUSIONS
In summary, different concepts and technologies have been reported in the literature
offering great prospects for the future application of lignin. However, there has been
limited investment in the development of a lignin-based industry , mostly due to the lack
of a reliable bulk source of high quality material and economically viable processes to
justify the expenditure. The nature of lignin varies with different types of feedstock, both
in terms of quantity and quality; thus, specific pre-treatments are required for extraction
from individual plant species (Phitsuwan et al., 2013). With respect to lignin substituted
phenol-formaldehyde resins, it has been demonstrated that the source from which lignin
is derived and the method of extraction have a strong influence on its properties
(Doherty et al., 2011). So far, there is no universal technology that can overcome the
recalcitrance of any plant biomass utilized (Phitsuwan et al., 2013). It has been proposed,
that the solution will most probably rely on a combination of different chemical,
biochemical, physical and thermal methods (Thakur et al., 2014).
68
1.8 AIMS OF THE STUDY
This study is a part of ADMIT Bio-SuccInnovate project, which encompasses a holistic,

integrative approach to drive innovations, that enable lignocellulosic components of
dedicated non-food crops and agricultural residues to be refined into a range of
sustainable added-value products and markets. Other partners research is focused on
valorization of the cellulose and hemicellulose fractions. The hypothesis addressed here
is that combined steam explosion-organosolv and bioconversion processes can be
developed that will enable utilisation of the lignin components of lignocellulosic
feedstocks in an economically viable integrated biorefinery.
The main aim of this study is to identify a comprehensive range of processing

technologies for the generation of industrially useful forms of lignin with the aim of
encouraging commercialization of lignin and helping to advance this area of research.
This will involve characterisation of lignin extracted from grass and wood feedstocks and
analysis of these fractions in the context of composition and suitability for the production
of biomaterials. In addition, the potential for bioconversion will be tested with selected
microbial fungal and bacterial strains by investigating their capability to utilize lignin as a
growth substrate. Low molecular weight lignin break-down products will be
characterized to determine enzymatic degradation pathways and identify chemicals
with a commercial value. Insoluble lignin products will be also characterized to assess
their potential as a raw material for other bio-based products.
As a part of ADMIT BioSuccInnovate project, it is anticipated, that outcomes of this

research will be of strategic importance to industrial biorefineries and contribute to the
development of the European bioeconomy. Moreover, knowledge and understanding
generated in this project may also benefit academic research community, which has
active programmes in conversion of lignocellulose biomass.
69
Chapter 2
70
Chapter 2: EFFECT OF STEAM EXPLOSION ON THE EXTRACTION OF
LIGNIN FROM GRASS AND WOOD
2.1 ABSTRACT
Steam exploded fiber, was used to develop a two-stage process for the production of
new lignin fractions from Miscanthus giganteus, wheat straw, corn stover, pine and
wood. The main aim of this study was to compare a yield of lignin extracted from pre-
treated feedstocks to lignin extracted from non-pretreated materials, and discuss their
potential use in a biorefining context. Acid Insoluble Lignin (AIL) and Acid Soluble Lignin
(ASL) in the starting material was calculated based on method adapted from NREL/TP-
510-42618 (Sluiter et al., 2004).Lignin extraction was carried using formic acid/acetic
acid/water organosolv process. Extraction efficiency was calculated as % based on total
lignin content. Both organosolv and a combined steam explosion-organosolv process
showed high lignin extraction efficiency for grass and hardwood, yielding 67.23 % - 93.27
% of total lignin content. The formic acid/acetic acid/water based organosolv process
gave the highest lignin yields from Miscanthus giganteus steam exploded with acid Commented [AW[4]: Can’t really say increase –can compare
with other treatment
treatment (MG SE Acid). The final extraction efficiency was 93.27 %. Compared with
non-pretreated material, steam explosion with acid treatment significantly increased
lignin extraction yield in the case of corn stover (CS) (20 %) and willow (WW) (18 %) (p
<0.05). This indicated a big potential of the two-stage process for lignin recovery from
these types of biomass. Steam explosion under alkaline (SE Alk) conditions did not
increase extraction efficiency in most of the feedstocks. The only exception was seen in
case of corn stover (CS) revealing statistically significant increase in lignin extraction yield
compared with non-pretreated biomass (32 %) (p< 0.05). Organosolv and steam
explosion-organosolv processes showed very low suitability for lignin extraction from
pine. The maximum recovery achieved was only 29.57 % of total lignin content.
Keywords: • Steam explosion • Organosolv lignin • Grass • Wood• Lignocellulosic

Feedstock• Lignin extraction
71
2.2 INTRODUCTION
Biomass is currently a great and important source of renewable energy in agriculture-

based countries, because of the abundant supply and low cost. This resource can be
easily used in a more efficient way as a raw material in the chemical industry (Zakaria et
al., 2017).
After cellulose, lignin is the second most abundant natural polymer in the world. Its
occurrence and relative amount in biomass mostly depends on the plant species.
Currently, lignin interest in Europe as high-added value material for specific applications
and formulations is rapidly increasing (Maniet et al., 2017). As a natural and renewable
material with useful chemical and physical properties, obtained at reasonable cost, lignin
can potentially substitute any product currently sourced from petrochemical industry
(Watkins et al., 2015). A major challenge is that lignin is difficult to isolate and generates
very high amounts of solid residue compared to other components of lignocellulose.
Therefore, investigating the effects of different pre-treatment processes is required to
optimize the extraction process (Pandey and Kim, 2011).
The optimal pre-treatment process should have a low capital and operation cost. It
should also be effective on a wide range of lignocellulosic materials and result in the
recovery of biomass components in a useable form. Many pre-treatment methods have
already been studied and are still in development (Agbor et al., 2011). According to
reports in the literature, steam explosion is currently more environmentally friendly than
other processes (Stelte, 2013). It is a thermo-physical process which causes mechanical
destruction of lignocellulosic material by vapocracking and explosive decompression
(Maniet et al., 2017). Another method of pre-treatment is the organosolv process.
Organosolv was developed in the 70-80s as an efficient and sulphur-free method to
produce high-quality pulp (Constant et al., 2015). This fractionation technology
comprises a range of processes in which lignocellulosic biomass is treated with an organic
solvent and water, usually at elevated temperature (such as 105 oC). The most common
solvents used in the process include ethanol, methanol, acetone or organic acids
(Snelders et al., 2014). However, formic acid/acetic acid-based mixtures were observed
to be more efficient than alcohols for degradation of lignin and hemicellulose from
72
various lignocellulosic materials (Lehnen et al., 2002., Sun et al., 2004). Organic acids Commented [AS[5]: Any other references??
I added second.
give good extraction yields of hemicellulose and lignin (Snelders et al., 2014).
The European Project ‘’Biocore’’ aimed to create a lignocellulosic biorefinery for

sustainable processing of biomass using the innovative formic acid/acetic acid-based
organosolv process, developed by one of the project partners, Compagnie Industrielle
de la Matière végétale (CIMV), at pilot scale. The process has already been optimised
within the project for feedstocks such as wheat and rice straw, poplar or hardwood
residues (Snelders et al., 2014). Other feedstocks tested for lignin extraction based on
the CIMV process also include Miscanthus giganteus (Vanderghem et al., 2011).
However, this method was not tested on these feedstocks in combination with other
pre-treatments. Only one study focusing on the effect of steam explosion on lignin Commented [AW[6]: Not clear what is meant here- do you
mean in combination with pretreatment or-
extraction by the CIMV extraction process was reported for fescue (Maniet et al., 2017). Comparison with other lignin extraction methods?- need to make
clear
No other reported studies were identified for lignin extraction efficiency under
Combination was only tested on fescue
formic/acetic acid conditions followed by catalysed steam explosion. Therefore, more in-
depth knowledge of the effect of feedstock source on lignin extraction yield, using
different pre-treatment and extraction processes, will provide information which could
improve the efficiency of an integral lignocellulosic biorefinery.
The main purpose of the study described in this chapter was to develop new methods
for extraction of different lignin fractions, focusing on maximising extraction yield. In this
study, lignin was extracted from grass (Miscanthus giganteus, wheat straw, corn stover)
and wood (pine, willow) using the formic/acetic acid/water organosolv method with and
without a steam explosion pre-treatment in acid or alkaline conditions. It was
postulated, that combining these two processes will influence the chemical properties
of lignin fractions, thereby make the extraction process very efficient. The initial lignin
content in the starting material was measured using the Klason method adapted from
NREL/TP-510-42618 procedure (Sluiter et al., 2004) and the final extraction yield was
calculated. Results showing effect of the pre-treatment on lignin extraction yields were
compared and discussed in a context of biorefining of lignocellulosic biomass.
73
2.3 MATERIALS AND METHODS
All experiments and analyses presented in this study were carried out using materials
and methods described below. Unless otherwise stated, all plastic ware and commonly
used chemicals of the highest grade available were purchased from Fisher Scientific
Scientific (Lecicestershire, UK) or Sigma-Aldrich Company Ltd (Dorset, UK). Any glassware
used in the carried out procedures were of Duran or Pyrex brand. For all steps, purified
water, (Elga Purelab Classic UV, VWS UK Ltd, 18.2 mega ohm-cm resistivity at 25 °C) was
used unless otherwise stated.
2.3.1 FEEDSTOCKS
Miscanthus giganteus (MG), corn stover (CS), wheat straw (WS), pine (PN) and willow
(WW) were used as feedstocks in this study. Miscanthus giganteus was supplied by
Aberystwyth University, Gogerddan, SY23 3EE, UK and had been harvested in spring 2012
at the post-senescent stage. The material was air dried to < 15 % moisture, and milled
to < 100 mm. Corn stover (Zea Mays ssp) was supplied by Rothamstead Research Station,
UK, it had been oven dried to < 15 % moisture, and was also milled to < 100 mm. The Commented [AW[7]: Should mention moisture content of all
feedstocks
wheat straw (Triticum ssp) was supplied by R.J. Edwards Agricultural suppliers,
Mention moisture content of Miscanthus
Aberystwyth, UK, the material was air dried, baled and milled to < 100 mm. The moisture
content was < 15 %. Willow (Salix ssp) (endurance variety) was was supplied by
Rothamstead Research Station and had been harvested in January 2014. The material
was chipped (Jensen 540T, Germany) during harvest and was oven dried to < 10 % as
quickly as possible to stop fungi causing degradation to the material. Pine (Pinus ssp) was
kindly provided by local supplier, variety unknown. The material was chipped (Jensen
540T, Germany) and oven dried to < 10 %.
2.3.2 STEAM EXPLOSION
Steam explosion was conducted in Beacon (Biorefining Centre of Excellence, pilot scale
biorefinery; Aberystwyth Univsersity). In this study, 500 g DM of all biomass types were
firstly pre-treated by soaking with 1 % H3PO4 (% w/w) (acid treatment), aqueous solution
of 1 % Ca(OH)2 (% w/w) (alkaline treatment) and kept in the oven for 1 hour at 50 oC.
74
The wet solids were then loaded into pre-heated chamber, filled with steam to 2 bar
above experiment level and steam exploded at 10 bars for 5 minutes. Afterwards, the
sample was removed from the explosion vessel and cooled down. It was subsequently
mixed with 1 litre of water and filtered/strained through a muslin bag. The fibre was
stored frozen for further processing.
2.3.3 LIGNIN DETERMINATION IN STARTING MATERIAL

2.3.3.1 Samples preparation
Prior to analysis, non-pretreated and steam exploded materials were knife milled to <
2mm and then fractionated using sieves (355 x 250 μm) (adapted from Hames et al., Commented [AW8]: Check sizes
2008).
2.3.3.2. Lignin measurement in starting material

Acid Insoluble Lignin (Klason lignin) and Acid Soluble Lignin contents were determined
for all samples of each feedstocks (non-pretreated and steam exploded) following
NREL/TP-510-42618 procedures (Sluiter et al., 2004). 300 mg of materials were added
to tared, glass pressure tubes. 3 mL of 72 % sulphuric acid was added to samples, mixed
and incubated in a water bath at 30 oC for 60 minutes with stirring every 10 minutes.
Following incubation, 84 mL of deionized water was added to each sample (in order to
dilute acid concentration to 4 %) and all samples were placed into an autoclave for one
hour at 121 oC. After completion of the autoclave cycle, the hydrolysates were cooled
and vacuum filtered through previously weighed and muffle furnace- dried, filtering
crucibles. The crucibles and the Acid Insoluble Residues (AIL) were dried in an oven at
105 oC for 24 hours, cooled in a desiccator and weighed. In order to determine ash
remaining in the sample, the crucibles and acid insoluble residue were transferred back
to the muffle furnace at 575 oC for 24 hours, cooled in a desiccator and weighed again.
The hydrolysate liquor was used to determine Acid Soluble Lignin (ASL) by measuring the
absorbance of the samples at an appropriate wavelength with a UV-visible
spectrophotometer. The final calculations of the amount of AIL and ASL were carried out
as described by (Sluiter et al., 2004). Analyses were carried out with 3 biological
replicates.
75
2.3.4 LIGNIN EXTRACTION
In order to investigate the effect of catalysed steam explosion on lignin extraction

efficiency, all studied feedstocks were subjected to the formic/acetic acid-based
organosolv process. The optimal conditions were determined based on literature
search. In this study, extraction processes were carried out using two stages: incubation
for one hour at 60 oC using a mixture formic acid/water and 3 hours refluxing at 107 oC
with a mixture of acetic acid/formic acid as they were optimal conditions earlier
reported by Mire et al., (2005). Prior to processing, samples were oven dried to 10 %
moisture content and milled to < 2mm. All samples extractions were carried out with a Commented [AS[9]: Method picked upbased on lit, dont write
the heating mantels, just write an initial study was done to verify if
reflux condenser attached to 250 mL round bottom flask (capacity 250 mL) and a heating the method works with different feedstocks . Good to say here that
you reviewed methods in literature and based on previous studies
you determined that these conditions described below were the
mantel. First trial was conducted with one replicate to verify if the method works with optimal for the formic/acetic process with your feedstocks
different feedtocks and didn’t involve constant mixing. Another trial was carried out with You don’t need to mention preliminary here as it does not involve
method development
3 replicates of which the lignin content was earlier measured (as described in section
All you need to say is that sample extractions were carried out with
2.3.3.2) and involved constant mixing. All biomass samples (10 g DM) were loaded into a relflux condensor attached to 250 mL round bottom flask and a
heating mantel.
the flasks and were treated with 75 mL of solvent mixture 60:40 (formic acid/water v/v) Write the first trial was done without mixing and the other
withmixing
for 1 hour at 60 oC. After that, 75 mL of solvent mixture 80:20 (acetic acid/formic acid
v/v) was added and all samples were refluxed for 3 hours at 107 oC. The final liquor ratio
was 40:40:20 (formic acid/acetic acid/water). The solid to liquid ratio used was 15:1
(w/v). After the refluxing, the slurry was filtered and washed with 150 mL of solvents
mixture 40:40:20 (formic acid/acetic acid/water) to separate the liquid from the solid
fraction. The cellulose fraction was washed with 150 mL of water to remove solid
residues and acids. In order to evaluate the minimum volume of water needed for lignin
precipitation, 10 mL of extraction liquor was taken and different amounts of water were
added to the sample (1:1, 2,5:1, 5:1, 8:1 v/v). The minimum ratio which achieved
effective precipitation of lignin was 2.5:1 (v/v) and this was used for subsequent lignin
precipitation. The mixture was centrifuged at 7000 min-1 for 15 minutes and the lignin
pellet was freeze-dried. Figure 2.1. shows the steps involved in lignin extraction and
recovery. Lignin fractions extracted from steam exploded material with acid treatment
and with alkaline treatment were coded, respectively: SE Acid, SE Alk. Lignin fractions Commented [AW10]: Should include untreated
extracted from non-pretreated materials were coded with feedstock names: MG lignin,
WS lignin, CS lignin, WW lignin, PN lignin.
76
Figure 4.181: Steps involved in the extraction and recovery of lignin by the organosolv process: 1) relflux
condensor with round bottom flask in heating mantle containing sample and extraction liquor, 2) lignin
precipitation after addition of water, 3) lignin pellet after centrifugation, 4) freeze-dried lignin extract.
2.3.5 REMOVING RESINS
The following procedure was carried out to remove resins from pine. Prior to processing,
samples were oven dried to a moisture content of 10 %. The procedure was conducted
in 500 mL conical flasks, following the method adapted from Foster et al., (2010). In order
to remove resins, 20 g of oven-dried steam exploded fibre as well as non-pretreated
material was extracted sequentially as follows: with 250 mL of 70 % of aqueous ethanol Commented [AW11]: In 250 mL flask? Not enough room- also
state whether at room temperature.
for 6 hours, with 250 mL of chloroform/methanol (1:1 v/v) for 12 hours and with 250 mL
acetone for 6 hours at room temperature, following constant agitation every 2 hours.
Between each step of the extraction, the material was vacuum-filtered with 250 mL of
70 % of aqueous ethanol, and the supernatant was discarded. Following the last, acetone
step, samples were left to dry overnight in the fume hood.
3.3.6 STATISTICAL ANALYSIS
The statistical calculations including Analyses of variance (ANOVA) and Tukey's tests
were performed using the computing software R (v. 2.14.0; R Development Core Team,
2005) at a 5 % significance level (P ≤ 0.05). Two-way ANOVA was used to test the factor
effect of treatment (SE Acid, SE Alk, non-pretreated) and lignin source (Miscanthus
giganteus, wheat straw, corn stover, pine and willow) on lignin content in starting
material and lignin extraction efficiency. Tukey's tests were used for multiple
comparisons between factor levels.
77
2.4 RESULTS
2.4.1 LIGNIN CONTENT IN STARTING MATERIAL
Total amount of lignin including Acid Insoluble Lignin (AIL) and Acid Soluble Lignin (ASL)
in each feedstock (non-pretreated and pre-treated) was measured based on the method
adapted from NREL/TP-510-42618 procedure (Sluiter et al., 2004) (as described in
section 2.3.3). The data for AIS, ASL and total lignin content were analysed by a two-way
ANOVA with replication followed by Tukey’s test. Table 2.1 shows the % lignin content, Commented [AW12]: need to carry out anova to determine
significant difference
on a dry matter basis, in extractive and starch-free grasses and wood samples. The
Commented [AW13]: Don’t generally call cereals - grasses
results of the analysis show, that total lignin content varied between different types of
feedstocks and with treatment. As seen in Table 2.1, the highest total lignin content in
non-pretreated material was observed for MG, with a content of 26.15 % and the lowest
total lignin content was observed for CS exhibiting only 18.33 %. Apart of MG lignin,
wood lignin fractions exhibited higher total lignin content than their grass counterparts.
Statistical analysis demonstrated, that total lignin content in CS was significantly different
from lignin content in PN and WW (p <0.05). In turn, lignin content in WS was significantly
different from lignin content in WW (p < 0.05) but not from lignin content in PN (p >0.05).
In comparison to non-pretreated samples, steam explosion with acid treatment
increased total lignin content in MG, WS, WW and PN. Based on results from Tukey’s
test, significant differences were only observed in case of WS and PN (p <0.05). In
contrast, the same feedstocks subjected to alkaline treatment showed decreased lignin
content, but the differences were not statistically significant (p >0.05). Among all grass
feedstocks, the highest total lignin content was observed for MG SE Acid (30.25 %) while
the lowest total lignin content was observed for non-pretreated CS, exhibiting 18.33 %.
In the case of wood feedstocks, the highest total lignin content was observed for PN SE
Acid (31.99 %) while the lowest value was observed for WW SE Alk lignin (23.88 %). All
grass lignin fractions exhibited lower ASL content following steam explosion with acid
and alkaline treatment. However, the differences in values were only significant for ASL
content in CS (p <0.05). In comparison, WW and PN subjected to steam explosion with
alkaline treatment revealed slightly higher ASL comparing to other treatment, but the
differences were not statistically different (p > 0.05).
78
Table 4.61: Lignin content in non-pretreated and steam exploded acid (SE Acid) and alkali (SE Alk) treated plant feedstocks. Data represent Acid Insoluble Lignin (AIL), Acid
Soluble Lignin (ASL) and Total Lignin as a percentage of dry matter (DM) with standard deviation (SD ±) of the mean (n=3). Values with the same letters within each lignin
fraction are not statistically different at p < 0.05 (based on Tukey’s comparison of means).
Lignin fraction (% DM)
Plant material Acid Insoluble Lignin (AIL) Acid Soluble Lignin (ASL) Total lignin
MG 25.67 ± 2.740 bc 0.48 ± 0.090 defg 26.15 ± 2.171 bc
MG SE Acid 29.95 ± 0.964 ab 0.30 ± 0.025 abcd 30.25 ± 0.797 ab
MG SE Alk 24.88 ± 2.915 bc 0.39 ± 0.031 bcdef 25.27 ± 2.390 bc
CS 17.26 ± 1.808 f 1.07 ± 0.077 i

18.33 ± 1.514 ef
CS SE Acid 17.65 ± 1.169 ef 0.34 ± 0.142 abcd 17.99 ± 1.049 f
CS SE Alk 18.55 ± 0.278 def 0.76 ± 0.050 h 19.31 ± 0.209 def
WS 18.72 ± 3.135 def 0.61 ± 0.018 gh 19.33 ± 2.546 def
WS SE Acid 27.85 ± 3.188 abc 0.35 ± 0.054 abcde 28.20 ± 2.559 abc
f fg f
WS SE Alk 17.99 ± 2.347 0.55 ± 0.118 17.56 ± 1.976
cd
WW 24.04 ± 2.295 0.45 ± 0.065 cdefg 24.49 ± 1.905 bcd
WW SE Acid 24.73 ± 0.800 bc 0.36 ± 0.023 abcdef 25.09 ± 0.662 bcd
WW SE Alk 23.35 ± 1.104 cde 0.53 ± 0.029 efg 23.88 ± 0.882 cde
PN 25.89 ± 0.418 bc 0.21 ± 0.010 ab 26.11 ± 0.348 bc
a
PN SE Acid 31.8 ± 0.539 0.19 ± 0.007 a 31.99 ± 0.444 a
PN SE Alk 25.33 ± 1.043 bc 0.27 ± 0.019 abc 25.60 ± 0.867 bc
79
2.4.2 LIGNIN EXTRACTION
2.4.2.1. Application of the organosolv procedure for extraction of lignin from a range of
feedstocks with different pre-treatments
Extraction of lignin from different feedstocks used in this study was carried out using the
formic acid/acetic acid organosolve procedure (as described in section 2.3.4). A Commented [AW14]: You don’t need to say this- can say
preliminary exp. Just to check procedure with different feedstocks
preliminary experiment was carried out to test the procedure with different feedstocks.
As shown in Figure 2.2. lignin extracts from non-pretreated samples have a range of
different colours, depending on the starting material. WS and MG lignin appears dark
brown in colour, CS lignin dark grey while PN and WW lignin are light brown and dark red
in colour, respectively. There was no correlation between the lignin colour and
treatment used prior to extraction. Tables 2.2 and 2.3 show lignin extraction yield data
as a percentage of dry matter. Results showed, that the highest lignin extraction yield
from grasses was obtained following steam explosion with acid treatment. In turn, the
lignin extraction yield following steam explosion with alkali treatment was very low. MG
SE Acid sample yielded the highest lignin extract with a value of 15.74 % , followed by
WS SE Acid and CS SE Acid with lignin yields of 12.51 % and 8.34 %, respectively. The
same pattern was observed for MG, WS and CS lignin extracted from steam exploded
material with alkaline treatment, yielding respectively only 8.96 %, 7.42 % and 6.87 %
(Table 2.2). In comparison with grasses feedstocks, hardwood and softwood subjected
to a pre-treatment process showed slightly different results. The lignin extraction yield
from willow increased following steam explosion with acid and alkaline treatment. WW
SE Acid yielded the highest lignin extract of 10.0 % , followed by WW SE Alk and non-
pretreated WW with lignin yields of 8.61 % and 6.64 % , respectively. In turn, steam
explosion under acidic and alkaline conditions resulted in decreased lignin extraction
yield from pine, achieving only 4.73 % and 2.91 %, respectively (Table 2.3). This
experiment demonstrated, that the organosolv process could be applied to these
feedstocks. In order to confirm lignin extraction efficiency, the experiment was repeated
with the same samples in triplicate and was carried out under the same conditions (as
described in section 2.4.3).
80
Figure 4.192: Organosolv extracted lignin fractions from a range of non-pretreated feedstocks:
1) Miscanthus giganteus (MG), 2) Corn stover (CS), 3) Wheat straw (WS), 4) Willow (WW), 5) Pine (PN).
Table 4.72: Organosolv lignin fractions extracted from range of grass feedstocks: Miscanthus giganteus
(MG), Wheat Straw (WS), Corn stover (CS) following steam explosion of acid, alkali and non-pretreated
material. Data represent lignin yield as a percentage of dry matter (DM).
Lignin extracted from steam exploded and non-pretreated material (% DM)
Plant material
Acid treatment Non-pretreated Alkali treated
MG 15.74 12.53 8.96
WS 12.51 9.00 7.42
CS 8.34 7.13 6.87
81
Table 4.83: Organosolv lignin fractions extracted from two wood feedstocks, Pine (PN) and Willow
(WW) following steam explosion of acid, alkali and non-pretreated material. Data represent lignin yield
as a percentage of dry matter (DM).
Lignin extracted from steam exploded and non-pretreated material (% DM)
Plant material
Acid treated Non-pretreated Alkali treated
WW 10.0 6.64 8.61
PN 4.73 5.75 2.91
2.4.2.2 Recovery of lignin from a range of feedstocks with different pre-treatments

following extraction of lignin by the organosolv procedure
The preliminary experiment in section 2.4.2.1. established, that the formic acid/acetic
acid organosolv procedure for lignin extraction was effective with the range of
feedstocks investigated. The procedure was repeated with the same feedstocks in
triplicate to estimate the total lignin recovery using this method (as described in section
2.3.4). The data for lignin extraction yield as % of DM were analysed by a two-way
ANOVA with replication followed by Tukey’s test. Table 2.4. shows the % lignin yield
calculated on a dry matter basis from 3 replicates. The results of the experiment mostly Commented [AW[15]: Need to carry out Anova (or other
suitable stats) to show significant differences
confirmed the outcomes for lignin extraction from grass feedstocks in the preliminary
test in terms of ranking, however over all yields were markedly higher. For grass samples,
the highest lignin extraction yield was obtained following steam explosion with acid
treatment. MG SE Acid revealed the highest lignin extraction yield of 28.29 % followed
by WS SE Acid with lignin yields of 19.0 %. Based on results from Tukey’s test, significant
increase in lignin extraction yield compared to non-pretreated material was only
observed in case of MG (p <0.05). In turn, lignin extraction yield following steam
explosion with alkali treatment was very low. The recovery of lignin from MG SE Alk and
WS SE Alk significantly decreased compared to non-pretreated material (p <0.05)
yielding 13.75 % and 9.76 % respectively. In the case of CS, the highest lignin extraction
yield was observed for samples steam exploded following alkali treatments: 16.78 % .
Compared to non-pretreated material, an increase in lignin extraction yield from CS SE
82
Alk was significantly different (p <0.05). Following the acid pre-treatment lignin recovery
from CS also increased (13.47 %), however the differences were not statistically
significant (p> 0.05). With respect to wood feedstocks, the highest yield was observed
for lignin extracted from hardwood as noted in the previous experiment. As previously
observed, steam explosion with acid treatment increased lignin yield from willow,
reaching 16.90 % . In turn, the lignin yield from WW SE Alk was 11.38 %. That was slightly
lower than in the case of non-pretreated feedstock. However, statystical analysis
determined, that differences in lignin extraction yield from pre-treated and non-treated
materials were not significant (p >0.05). The lowest extraction yield was observed for
lignin extracted from softwood. Following steam explosion under acidic and alkaline
conditions lignin extraction yield decreased respectively to 5.46 % and 3.38 %. Such a
decreasing pattern was in accordance with results obtained in the preliminary
experiment. However, based on results from Tukey’s test, significant decrease in lignin
extraction yield compared to non-pretreated material was only observed in case of lignin
extracted from PN SE Acid (p< 0.05) (Table 2.4).
83
Table 4.94: Organosolv lignin fractions extracted from a range of grass and wood feedstocks: Miscanthus
giganteus (MG), Wheat Straw (WS), Corn stover (CS), Pine (PN), Willow (WW) following steam explosion
of acid (SE Acid), alkaline (SE Alk) and non-pretreated material. Data represent lignin yield as a
percentage of dry matter (DM) with standard deviation (SD ±) of the mean (n=3).
Plant material Extracted Lignin (% DM)
MG 18.62 ± 0.109 d
MG SE Acid 28.29 ± 3.901 ab
MG SE Alk 13.75 ± 0.951 ef
CS 10.02 ± 0.470 f
CS SE Acid 13.47 ± 1.739 ef
CS SE Alk 16.78 ± 1.280 de
WS 14.93 ± 1.007 de
WS SE Acid 19.0 ± 0.641 d
WS SE Alk 9.76 ± 0.355 f
WW 12.16 ± 2.421 bc
WW SE Acid 16.90 ± 2.710 bc
WW SE Alk 11.38 ± 1.055 c
PN 7.72 ± 0.444 bc
PN SE Acid 5.46 ± 0.247 a
PN SE Alk 3.38 ± 0.070 bc
84
2.4.3 LIGNIN EXTRACTION EFFICIENCY
Table 2.5 summarizes the % lignin extraction efficiency, based on total lignin content in Commented [AW[16]: Need Anova or other method to show
significant difference
the starting material (as presented in section 2.4.1). As seen in Figure 2.3 the highest
lignin extraction efficiency reaching 93.27 % was observed for MG subjected to steam
explosion with acid treatment. The same pattern was observed in the case of WW SE
Acid, which exhibited the highest lignin yield following pre-treatment under acid
conditions: 67.23 %. Comparing to non-pretreated materials, an increase in lignin
extraction yield from both types of feedtocks steam exploded with acid treatment was
statistically different (p < 0.05). Although, the amount of lignin extracted from CS SE Acid
was higher than in case of non-pretreated material, steam explosion with alkali
treatment showed the best extraction efficiency, significantly increasing lignin yield to
86.89 % (p <0.05). The amount of lignin extracted from MG SE Alk, and WW SE Alk was
significantly lower compared to feedstocks steam exploded with acid treatment (p< 0.05)
but not significantly different compared to non-pretreated feedstocks (p> 0.05). The final
recovery yield reached 54.60 % and 47.56 %, respectively. The highest lignin yields for
WS and PN feedstocks were observed for non-pretreated materials, with values of 78.73
% and 29.57 % respectively. Following steam explosion with acid and alkali treatment,
total lignin extraction efficiency from WS decreased to 67.78 % and 56.16%, respectively.
Based on results from Tukey’s test, signicant decrease in lignin extraction yield
comparing to untread material was only observed in case of lignin extracted from WS SE
Alk (p < 0.05). In turn, the amount of lignin extracted from PN under acid and alkaline
conditions decreased to 17.06 % and 13.23 %, respectively, but the differences were not
statistically different (p> 0.05). Low extraction yields for lignin extracted from pine
indicated, that the organosolv and combined steam explosion-organosolv process
investigated here is not suitable for this type of feedstock. Pine was further investigated
and the results shown in section 2.4.4.
85
Lignin extracted as % of total lignin
120.00
Lignin extracted as % of total lignin
100.00
80.00
60.00
40.00
20.00
0.00
MgAcid Mg MgAlk CSAcid CS CSAlk WSAcid WS WSAlk PineAcid Pine PineAlk WWAcid WW WWAlk
Lignin
Figure 4.203: Efficiency of lignin extraction from a rage of grass and wood feedstocks: Miscanthus giganteus (MG), Wheat Straw (WS), Corn stover (CS), Pine (PN), Willow
(WW) following steam explosion of acid (SE Acid), alkaline (SE Alk) and non-pretreated material. Data represent lignin yield as a percentage of total lignin content in starting
material with standard deviation (SD ±) of the mean (n=3).
86
Table 4.105: Efficiency of Organosolv lignin extraction process with a range of different grass and wood
feedstocks: Miscanthus giganteus (MG), Wheat Straw (WS), Corn stover (CS), Pine (PN), Willow (WW)
following steam explosion of acid (SE Acid), alkaline (SE Alk) and non-pretreated material. Data represent
lignin yield as a percentage of total lignin content in starting material with standard deviation (SD ±) of
the mean (n=3). Values with the same letters are not statistically different at p < 0.05 (based on Tukey’s
comparison of means).
Plant material Lignin extracted as % total lignin
MG 71.68 ± 5.964 bcd
MG SE Acid 93.27 ± 8.026 a
MG SE Alk 54.60 ± 2.006 de
CS 54.86 ± 2.870 de
CS SE Acid 74.74 ± 4.580 bc
CS SE Alk 86.89 ± 4.782 ab
WS 78.73 ± 7.334 abc
WS SE Acid 67.78 ± 4.779 cd
WS SE Alk 56.16 ± 4.941 de
e
WW 49.40 ± 4.739
WW SE Acid 67.23 ± 7.464 cd
WW SE Alk 47.56 ± 1.867 e
PN 29.57 ± 1.090 f
PN SE Acid 17.06 ± 0.480 f
PN SE Alk 13.23 ± 0.324 f
87
2.4.4 METHOD DEVELOPMENT FOR LIGNIN EXTRACTION FROM SOFTWOOD
Results presented in section 2.4.3 showed, that lignin extraction yield from pine was very
low. In order to investigate whether removing resins increases the lignin extraction
efficiency from pine, an experiment was carried out to investigate the effect of removing
extractives from pine on lignin extraction with the organosolv process. Resins were
removed as described in section 2.3.5. Lignin extraction was carried out according to the
protocol presented in section 2.3.4. Table 2.6. shows lignin extraction yield as a % of dry
matter. Results of the experiment revealed, that lignin extraction efficiency from
extractives-free biomass was lower than in case of non-pretreated feedstock. This
indicates that the combined steam explosion-organosolv process investigated here is not
suitable for this type of feedstock.
Table 4.116: Organosolv Pine (PN) lignin fractions extracted from extractives-free (EF) and non-
pretreated biomass. Data represent lignin yield as a percentage of dry matter (DM).
Plant material Extracted Lignin (% DM)
PN 3.4
PN SE Acid 2.94
PN SE Alk 3.37
PN (EF) 2.9
PN SE Acid (EF) 1.83
PN SE Alk (EF) 1.29
88
2.5 DISCUSSION
Assessing the chemical composition of lignocellulosic biomass, is a necessary step for the
optimisation of biorefining approaches (Collins et al., 2014). Acid Insoluble Lignin (AIL)
and Acid Soluble Lignin (ASL) content measured according to NREL/TP-510-42618 (Sluiter
et al., 2004) is one of the most common methods for determining lignin content in
feedstocks. As part of this study, total lignin content was measured in all non-pretreated
and steam exploded feedstocks prior to extraction. Chosen feedstocks were subjected
to steam explosion under acid and alkaline conditions for 5 minutes at 10 bars and 185
°C, which were optimised in the Bio-SuccInovate project. Alkali pre-treatment with
calcium hydroxide was chosen as it is less expensive, safer than sodium hydroxide Commented [AS[17]: Not sure about revovering carbon
hydroxide check
(Maurya et al., 2015., Chang et al., 2017) and could be easier recovered as insoluble Have checked and few papers seem to mention these
calcium carbonate by reaction with carbon dioxide (Mosier et al., 2005; Soares Rodrigues
et al., 2016). Although acid-based steam explosion is usually carried out with sulphuric
acid, because of the low-cost and easy availability (Wang et al., 2011), it is corrosive to Commented [AW[18]: GOOD REF FOR PHOSPHORIC USE-
OPTIMIZATION OF DILUTE-PHOSPHORIC-ACID STEAM
PRETREATMENT OF EUCALYPTUS BENTHAMII FOR BIOFUEL
metals. For this reason, phosphoric acid which is less harsh on equipment (Castro et al., PRODUCTION
2014) was used instead. As predicted, the total lignin content in raw wood feedstocks
was higher compared to most of the grass samples (Kumar et al., 2009). Non-pretreated
PN and WW exhibited the lignin content with values of 26.11 % and 24.49 % on a DM
basis, respectively. For grasses, the lignin content reported in non-pretreated CS was
18.33 % on a DM basis and statistically differed from lignin content in non-pretreated PN
and WW (p< 0.05). In turn, the total lignin content in WS revealing 19.33 % on a DM
basis significantly differed from lignin content in PN (p< 0.05), but not from lignin content
in WW (p> 0.05). Although, the lignin content in MG (26.15 % on a DM basis) was higher
than lignin content in wood feedstocks, the differences were not statistically different
(p> 0.05) (Table 2.1). These results were in close agreement with data published in the
literature. Lignin contents of 25 % for willow and 26 % for pine was previously observed
by Sandak et al., (2017) and Waliszewska et al., (2015). Brosse et al., (2012) reported a Field Code Changed
similar lignin content of 25 % for Miscanthus giganteus. Lignin contents of up to 18 % for

CS and WS were previously observed by Templeton et al., (2009) and del Río et al.,
(2012), respectively. Although values observed in the current study are mostly consistent
with lignin contents reported in previous studies, differences in lignin content within
89
feedstocks are very common. The composition can vary according to agronomic
conditions, location and climate in addition to heritable variation (Collins et al., 2014). It Commented [AW[19]: Chapter 1Compositional Analysis
of Lignocellulosic Feedstocks. 1. Review and Description
of Methods
should be noted, that the presence of protein can cause an over-estimate of lignin by Justin B. Sluiter, Raymond O. Ruiz, Christopher J.
Scarlata, Amie D. Sluiter, and David W. Templeton*
the Klason method and the grass samples are likely to contain some protein (Sluiter et
al., 2010).
The lignin analysis showed that total content varied not only between feedstocks but
also between treatments. Total amount of lignin determined in MG, WW, WS and PN
steam exploded with acid treatment was higher than determined for non-pretreated
materials. Determination of Klason lignin steam exploded with acid treatment revealed
an 0.4 % to 9 % increase in lignin content (Table 2.1). However, statistical analysis
demonstrated, that significant differences between the treatments were only observed
in case of WS and PN (p< 0.05) (Table 2.1). It is very probable, that degradation products
of carbohydrates (such as furfural), acids or extractives could condense with lignin during
steam explosion, which has been previously reported by Wildschut et al., (2013).
Condensation of lignin with proteins under acid conditions could also occur during steam
explosion (Whitmore, 1982). Although, the generation of so called ‘pseudolignin’ has
been proposed by many researchers, there is still a lack of understanding of the
fundamental chemistry surrounding this phenomenon (Hu et al., 2012). In turn, Field Code Changed
feedstocks subjected to alkali pre-treatment revealed an 0.6 % to 0.8 % decrease in

Klason lignin content compared with non-pretreated materials but the differences were
not statistically significant (p< 0.05) (Table 2.1). It’s very likely, that lower Klason lignin
content could be a result of lignin solubility during steam explosion in the presence of Commented [AS[20]: Maybe better to give average amount
lower than untreated as %
Ca(OH)2. Lignin solubility during steam explosion in alkaline conditions is very efficient.
However, it has very little effect on solubilisation of hemicellulose and cellulose. Results
presented by Dawson and Boopathy, (2008)., Singh and Bishnoi, (2013)., and Monlau et
al., (2012) revealed, that alkali pre-treatments effectively remove a high proportion of
the lignin in comparison with other pre-treatment technologies. Based on results
reported by David Walker (Aberystwyth University), steam explosion with alkali Commented [AS[21]: Name person
treatment exhibited very low sugar removal and a high content of lignin degradation
products in the hydrolysate fraction. This is consistent with previous studies. It is of
interest, that compared to other feedstock, CS following steam explosion with alkali
90
treatment exhibited the highest Klason lignin content (Table 2.1). That would indicate,
that CS subjected to pre-treatment under alkaline conditions does either not solubilise
lignin, or that ‘pseudolignin’ is created instead. However, comprehensive analysis of the
solid lignin and hydrolysate fractions is necessary to determine the factors accounting
for the increase in Klason lignin in alkaline conditions.
In order to investigate the effect of catalysed steam explosion on lignin extraction Commented [AW[22]: It would be worth including a few
comparisons with other methods of lignin extraction-
efficiency, all studied feedstocks were subjected to the formic/acetic acid-based
Project partners
organosolv process. Currently, organosolv an ionic liquids are two methods generating
Soemthign about sulfur-free, mention ionic liquid
lignin with useful physico-chemical properties. Organosolv and ioninc liquid lignin
fractions are unique from the rest of the lignin fractions due to their low molecular
weight, high purity, and the ease of dissolution in certain solvents. However, ionic liquid
fractionation has the challenge/limitation of scalability due to high cost of ionic liquids
and their use still needs to be explored (Espinoza-Acosta et al., 2014). Taking into account
that CIMV, one of the project partners has already optimised formic/acetic acid
organosolv process for lignin extraction from wheat straw it has been postulated, that
subjecting other non-pretreated and pre-treated feedtocks to this type of extraction
could play an important role in creating integral lignocellulosic biorefinery. In the current
study, organosolv pulping consisted of two stages: incubation for one hour at 60 oC using
a mixture formic acid/water and 3 hours refluxing at 107 °C C with a mixture of acetic
acid/formic acid as they were optimal conditions earlier reported by Mire et al., (2005).
It has been suggested, that the impregnation stage in formic acid plays a key role in
delignification process. Moreover, increasing formic acid concentration in the refluxing
stage can even improve the delignification rate (Mire et al., 2005). Preliminary
experiment results indicated, that lignin extraction efficiency significantly increased in
the case of feedstocks steam exploded with acid treatment. In turn, steam exploded
materials under alkaline conditions subjected to the organosolv process showed lower
extraction yields compared to non-pretreated materials (Tables 2.2 and 2.3). When the
process was repeated with the same feedstocks in triplicate, results showed a similar
pattern to that observed in the preliminary experiment however over all yields were
Commented [AW[23]: It would be good to try and explain
markedly higher. (Table 2.4). The reason behind this is, that during second trial the improved yield- something about obtimising the process- anything
different that you did
pulping process was carried out with constant agitation. The bigger accessibility of
Write that resukts were higher than preliminary
Write in discussion why for example because of mixnign
91
biomass to the solvents could potentially influence the final extraction yield. It’s also
worth to add, that comparing to preliminary extractions carried out separately using
basic lab equipment, second trial was performed for all grass and wood feedstocks at the
same time at pilot-scale facilities. This allowed for more constant conditions, thereby
efficient extraction. The final extraction efficiency was calculated based on the % of total
lignin content. Out of the five different feedstocks with different pre-treatments,
organosolv extraction of MG SE Acid achieved the highest extraction efficiency with an
increase of 21 % compared with non-pretreated material and a yield of 93.27 % of total
lignin content. Combined steam explosion acid based-organosolv procedure also
increased the extraction efficiency in case of CS (20 % increase; 74.74 % yield), and WW
(18 % increase; 67.23% yield) (Figure 2.3). Based on results from Tukey’s test, the
differences in lignin extraction yield from non-pretreated and steam exploded materials
under acidic conditions were statistically significant (p< 0.05) (Table 2.5). The reason for
this is, that during steam explosion lignin is softened and released from plant cell wall
(Stelte, 2013) thereby making the extraction process more efficient. In contrast,
organosolv extraction of steam exploded materials with alkali treatments did not
improve extraction efficiency with most of the feedstocks (Figure 2.3). In case of WS,
significant decrease of 23 % compared with non-pretreated material was observed (p <
0.05). In turn, a decrease of 17 % was also reported for lignin extraction from WW and
PN but the differences in values were not statistically different (p> 0.05) (Table 2.5).
Lower lignin extraction in case of MG, WS and PN pre-treated with steam explosion
under alkaline conditions indicated, that such a sort of pre-treatment is unsuitable for
lignin recovery from these types of feedstocks. However, it is interesting that extraction
of lignin from CS steam exploded under alkaline conditions showed significantly higher
lignin recovery comparing to non-pretreated material (p< 0.05) with a yield of 86.89 %
of total lignin content. The final extraction yield was also higher comparing to lignin
extracted from CS SE Acid, but the differences between the treatments were not
statistically different (p> 0.05). None of the pre-treatments resulted in an increase in
lignin extraction yield in the case of PN and WS, indicating that for good rate of lignin
recovery, non-pretreated material is the optimal starting material (Table 2.5/Figure 2.3).
In this study, extraction yield of lignin from WS exhibited 79 % of total lignin content,
which was in close accordance with data presented by Snelders et al., (2014) who
92
showed, that 84 % of total lignin could be recovered from non-pretreated WS, using the
formic/acetic acid-based organosolv process. However, it should be noted, that the ratio
of acetic acid/formic acid used for delignification in study presented by Snelders et al.,
(2014) reached 65:35 (v/v), which was higher compared to the ratio used in this
research: 40:40 (v/v). Xu et al., (2006) who already studied the effect of formic acid on
delignification in wheat straw concluded, that obtained pulp still contained some lignin,
hemicellulose and ash. The higher recovery obtained by Snelders et al. (2014) may
indicate, that the increased ratio of acetic acid to formic acid, is a key factor for high
lignin recovery. However, it is also possible that some of the lignin is solubilized during
pulping process, which might decrease the final extraction yield. Preliminary study of Commented [AW[24]: Did you analyse the liquor for lignin
products?
organosolv liquor carried out by David Walker (Aberystwyth Univeristy) already exhibited
Mention about future work for analysis this fraction
the presence of lignin degradation products such as vanillin or syringaldehyde. However,
further and more comprehensive analysis would need to be carry out to fully confirm
this theory. Explaining such changes is very difficult, taking into account a range of
different factors. The extraction efficiency can be influenced by time, temperature or
even particle size (Zakaria et al., 2017). Both organosolv and combined steam explosion-
organosolv process showed a high lignin extraction efficiency for grass and hardwood
feedstocks, yielding 67.23 % - 93.27% of total lignin content. In turn, the results obtained
for lignin extraction yield from pine indicated, that such processes are poorly suited for
softwood feedstocks. Taking into account different treatments, the maximum lignin
recovery achieved from from pine was only 29.57 % of total lignin content (Figure
2.3/Table 2.5).
As previously reported by CIMV, softwood was initially excluded from the process,
because the resin content is a serious handicap, hampering the penetration of the
solvent system (BIOCORE, 2014). In order to investigate the effect of resins on lignin
extraction from pine, the process was carried out on extractives-free biomass and
compared to non-pretreated material. The results of analysis showed, that there was no
positive correlation between resins removal and lignin extraction efficiency. Moreover,
the final lignin yield extracted from extractives-free biomass was lower compared to non-
pretreated feedstocks (Table 2.6). That was most likely because some lignin has already
been removed with extractives. Similar results were also reported by Burkhardt et al.,
93
(2013). In her study, prior removal of extractives had a substantial effect on the
compositional analysis of the forest residues and the determined lignin content
decreased significantly (3-18 %). Above outcomes confirmed, that organosolv and
combined steam explosion organosolv procedure is not suitable for lignin extraction
from softwood materials. Softwood lignin with a higher content of guaiacyl units, is not
easily degraded by acids. Guaiacyl lignin restricts fibre swelling and enzymatic
accessibility more than hardwood lignin which has a higher content of syringyl groups.
Thus, finding methods for effective pre-treatment of softwoods is currently an
important issue (Garcia-Cubero et al., 2010).
Although both steam explosion and organosolv extraction processes have advantages
and disadvantages, they suit the biorefinery concept where all the components of
biomass can be recovered, valorised and applied to a range of different applications.
Within the project, the proposed two-step process has been developed to add value to
steam exploded fibre residue for the recovery of the hemicellulose fraction. As shown
above, processing dedicated energy crops using different pre-treatments combined with
the formic/acetic organosolv method, generated an array of different lignin types and
affected the final extraction yield. Taking into account the fact that cultivation of
dedicated energy crops expands in order to meet government mandates for renewables,
the available feedstocks for the production of lignin-derived products will increase as
well (Ten and Vermerris, 2015). However, further research is needed to characterise
obtained lignin fractions, identify potential bio-based products and make them more
competitive than those obtained from the petroleum industry.
94
2.6 CONCLUSIONS Commented [AW[25]: You need to rewrite this section- don’t
include data- just overall observations and conclusions-
Eg. Organosolve good for grasses and hardwood- evidence that acid
causes pseudolignin formation and alkali probably solubilises lignin-
The results presented in this study showed, that lignin content and the final extraction conclusion suitable method for lignin extraction from grass and
hardwood feedstocks.
yield depends on the type of feedstock and pre-treatment. The compositional analysis
of studied materials revealed, that in most cases lignin content in wood feedstocks was
higher comparing to grasses. Steam explosion with acid treatment increased the total
lignin content, indicating possible presence of ‘’pseudolignin’. Lower lignin content
following steam explosion under alkaline conditions highlighted the fact, that some lignin
already solubilized during the pre-treatment with CaOH2. Both organosolv and combined
steam explosion-organosolv process revealed a high lignin extraction efficiency for grass
and hardwood feedstocks. Steam explosion with acid treatment significantly increased
lignin extraction yield in case of MG, CS and WW showing, that two-stage process has
the biggest potential for lignin extraction from these type of feedstocks. In turn, very low
extraction efficiency in case of PN indicated, that both processes are poorly suited for
softwood and more work needs to be carried out to fully enable its potential.
95
Chapter 3
96
Chapter 3: COMPARATIVE STUDY ON THE EFFECT OF PRE-TREATMENT
WITH STEAM EXPLOSION ON CHARACTERISTICS OF LIGNIN
FROM GRASS AND WOOD
3.1 ABSTRACT
Lignin from a range of grass: Miscanthus giganteus, wheat straw, corn stover and wood:
willow, pine feedstocks, extracted using combined steam explosion-organosolv process
was compared to organosolv lignin extracted from non-pretreated feedstocks. The main
aim of this study was to characterise all lignin fractions in terms of composition and
suitability for the production of high-value products. Lignin was characterized by their
composition and solubility in organic solvents. Physical properties such as thermal
stability and molecular weight were measured respectively by TGA and SEC. Additionally,
spectroscopy methods such as FTIR and 31P-NMR were implemented to analyse
functional groups. Results showed, that all lignin samples were relatively pure (84 %
Klason lignin, < 4.8 % of carbohydrates and 5 % of ash). The high amount of total OH
groups, aliphatic groups and high solubility in dioxane and DMSO observed with lignin
extracted from alkali treated, steam exploded Miscanthus giganteus (MG SE Alk lignin),
alkali treated, steam exploded wheat straw (WS SE Alk lignin), non-pretreated wheat
straw (WS lignin), non-pretreated Miscanthus giganteus (MG lignin), non-pretreated
pine (PN lignin) and acid treated, steam exploded pine (PN SE Acid lignin) indicated their
high potential for use in polyurethanes. The low molecular weight and polydispersity
observed in the case of lignin extracted from acid treated, steam exploded wheat straw
(WS SE Acid lignin), non-pretreated corn stover (CS lignin) and wheat straw (WS lignin),
acid and alkali treated, steam exploded pine (PN SE Acid and PN SE ALk lignin,
respectively) and alkali treated, steam exploded willow (WW SE Alk lignin) could favour
incorporation into phenol-formaldehyde resins (PF resins). The broad range of good
properties of lignin extracted from acid treated, steam exploded wheat straw and pine Commented [AS[26]: WS lignin had all above properties (good
purity, low molecular weight etc
(WS SE Acid lignin and PN SE Acid lignin, respectively ) indicated, that it might be a
potential source as a functional ingredient of polyesters, polyurethanes and PF resins.
Keywords: • Grass • Wood • Lignin composition • TGA• FTIR • SEC • Solubility •.31P-
NMR • High-value • Products
97
3.2 INTRODUCTION
Lignocellulosic biomass, including energy crops and agricultural waste are renewable
resources with significant potential. Lignin, which is one of its major components, has
been traditionally viewed as a 'waste product' in paper and biofuel production (Lange et
al., 2013). However, its chemical structure indicates that it might be a potential
intermediate for the production of commercial products such as polymers or resins
(Cotana et al., 2014).
Lignin is the most abundant aromatic biopolymer in nature, mainly composed of

phenylpropane units. They are synthesized from three aromatic alcohol precursors
(monolignols); i.e., p-coumaryl, coniferyl and sinapyl alcohol. These cross-linked phenolic
substructures give rise to residues defined as p-hydroxyphenyl (H, from coumaryl
alcohol), guaiacyl (G, from coniferyl alcohol) and syringyl (S, from sinapyl alcohol)
(Huijgen et al., 2014).
Large amounts of lignin are produced yearly by the paper and pulp industry, as a
byproduct of delignification. Due to the development of new technologies to produce
second-generation biofuels and chemicals from lignocellulosic biomass, the availability
of lignin is forecasted to increase significantly (Boeriu et al., 2014). To meet the goal of
replacing 30 % of fossil fuels by biofuels by 2030, approximately 227 hm3 of bioethanol
would be required, which would generate about 0.225Gt of lignin (Sahoo et al., 2011).
Development of value-added bio-products is considered a high priority in order to

achieve a sustainable bioeconomy (Cotana et al., 2014). Direct use of lignin or conversion
to lower-molecular weight aromatic, monomeric building blocks for polymer production
can create a commercial opportunity (Lange et al., 2013). The most promising
commercial applications of macromolecular lignin include carbon fibres, polymer
modifiers (for example utilising lignin as high value copolymer additives) or adhesives
and resins (for example for formaldehyde-free applications) (Zakzeski et al., 2010). In
turn, aromatic building blocks derived from lignin can be further converted into a variety
of useful chemicals. Lignin-derived chemicals are also potential major precursors for
plastics, resins and other polymer materials (Jongerius, 2013).
98
There are currently two identified potential high volume, added-value applications for
lignin utilisation, namely polyurethane and phenol-formaldehyde resins-based materials.
Polyurethanes are among the most versatile synthetic materials and they are
incorporated in many consumer products including: insulating panels, shoes (soles),
mattresses, toys or kitchen sponges (BIOCORE, 2014). In 2016, with a global production
of 18 Mt, PUs rank 6th among all polymers based on annual worldwide production
(Cornille et al., 2017). In turn, phenol-formaldehyde resins among others are used for
gluing wood based panels for outdoor and interior use (Tuomi and Mäkine, 2010).
Mostly, their use is as an adhesive and includes exterior-grade panels, e.g. cement mould
boards (Zhang et al., 2013). They can be also used for varnishes and billiard or pool balls
(Gosselink, 2011). The global demand for phenol-formaldehyde resins is more than 2.5
million tonnes per year (Tuomi and Mäkine, 2010).
Integration of technical lignin into multi-component materials has become an important

goal over the last 10-15 years. The main incentives are low cost, availability, abundance,
sustainability and other properties such as mechanical strength, good thermal
properties, chemical friction and water resistance. However, processes developed for
lignin-based polymer production at a research level have not been scaled up to an
industrial level yet (Laurichesse and Avérous, 2013). Therefore, investigating the
fundamental chemistry of lignin from different feedstocks and the effects of different
pre-treatment processes is a prerequisite to determine the optimal lignin source for the
production of lignin-derived components.
A currently applied technology is steam explosion which achieves separation of biomass

fibres by steam treatment followed by a sudden release of pressure to produce steam
explosion. (Stelte, 2013). As steam explosion will mainly make the fibre matrix more
accessible for conversion, an additional extraction step is needed to separate substantial
amounts of lignin. Pre-treatments including organosolv fractionation were developed as
a delignification technique to solubilise lignin and hemicellulose and use cellulose for
paper making (Cybulska et al., 2012). Studies have demonstrated, that organosolv lignin
can be usefully incorporated into construction and packaging materials. It can partially
substitute phenol in phenol-formaldehyde resin and studies have shown, that lignin
substituted resins can act as effective binders for oriented strand board panels (Donmez
99
Cavdar et al., 2008). Furthermore, organosolv lignin derivatives (modified with alkylene
oxides e.g propylene oxide) have been used to produce polyurethane and isocyanurate
resins (Lora and Glasser, 2002).
A lot of studies have been carried out on the analysis of lignin fractions generated by
organosolv process from grass (El Hage et al., 2009; Sun et al., 1997; Hu et al., 2012
Sammons et al., 2013) and wood feedstocks (Sammons et al., 2013; Nitsos et al.,2016;
Constant et al., 2016). With regard to grass feedtocks, the comprehensive analysis of
lignin fractions extracted by formic/acetic acid-based organosolv from wheat straw and
Miscanthus giganteus has already been reported by Snelders et al., (2014) and
Vanderghem et al., (2011), respectively. However, the effect of organosolv extraction on
chemical composition of lignin from corn stover and the effect of pre-treatments on
lignin properties has not been reported.In turn, the comprehensive analysis of lignin
fractions extracted by formic/acetic acid-based organosolv methods from wood
feedstocks has only been reported for hardwood (BIOCORE, 2014; Simon et al., 2014).
The effect of pre-treatments and the CIMV organosolv extraction process on chemical
composition of lignin from softwood has not been reported to date.
As seen in Chapter 2, organosolv and combined steam explosion-organosolv extraction

process already exhibited high potential for lignin recovery from grass and hardwood
biomass. In turn, the same process exhibited much lower feasibility for lignin recovery
from softwood. However, it was postulated that combining these two processes would
affect the chemical properties of lignin fractions, thereby the quality of potential lignin-
derived products. In this study, lignin fractions extracted from Miscanthus giganteus,
corn stover, wheat straw, pine and willow using the formic/acetic acid organosolv
method with and without a steam explosion pre-treatment in acid or alkaline conditions
were subjected to comprehensive analysis. Lignin characteristics were assessed by their
purity and solubility in organic solvents. Thermal stability and molecular weight were
measured respectively by Thermogravimetric analysis (TGA) and Size Exclusion
Chromatography (SEC). Analysis of functional groups was carried out by spectroscopy
methods including Fourier Transform Infrared Spectoscropy (FTIR) and Phosphorus-31
Nuclear Magnetic Resonance (31P-NMR). Furthermore, the relationship between
analytical characterisation data of the different lignin fractions and process conditions
100
has been studied. Results showing different properties of lignin were discussed in terms
of composition and suitability for production of polymers and resins.
All analyses presented in this study were carried out using materials and methods
described below. Unless otherwise stated, all plastic ware and commonly used chemicals
of the highest grade available were purchased from Fisher Scientific Scientific
(Lecicestershire, UK) or Sigma-Aldrich Company Ltd (Dorset, UK). Any glassware used in
the carried out procedures were of Duran or Pyrex brand. For all steps, purified water,
(Elga Purelab Classic UV, VWS UK Ltd, 18.2 mega ohm-cm resistivity at 25 °C) was used
unless otherwise stated.
3.3.1 RAW MATERIAL
Lignin fractions extracted from the following grass: Miscanthus giganteus (MG), corn
stover (CS), wheat straw (WS) and wood: willow (WW), pine (PN) biomass using
organosolv and combined steam explosion-organosolv process (as described in section
2.3.2, 2.3.4) were used as feedstocks in this study. Lignin fractions extracted from pre-
treated material included steam exploded material with acid treatment (SE Acid) and
with alkaline treatment (SE Alk). A sample of wheat straw lignin which was used as a
lignin standard was kindly supplied by Compagnie Industrielle de la Matière végétale
(CIMV) Pomacle, Champagnes-Ardennes, France. Kraft lignin, Alcell mixed hardwoods
lignin and other lignin standards were kindly supplied by Wageningen Food and Biobased Commented [AW[27]: Should give details e.g. Alcell lignin
Research.
3.3.2 LIGNIN ANALYSIS AND CHARACTERISATION

3.3.2.1 Lignin composition (purity)
Lignin fractions were treated with a two-step sulphuric acid incubation with 12M H2SO4
at 30 oC for 1 hour, followed by 1M H2SO4 at 100 oC for 3 hours (Gosselink et al., 2010).
The Acid Insoluble Lignin was determined after filtration and hot water washing over a
G4 glass fibre crucible (TAPPI method T 222 om-83, 1999). Acid Soluble Lignin was
spectrophotometrically determined at 205 nm (TAPPI useful method UM 250 um-83,
101
1991). For the measurement of sugar residues, the hydrolysate was neutralized by
calcium carbonate until pH ceased to be acidic as indicated by bromophenol blue. Commented [AW[28]: Any idea of pH range?
Resulting monosaccharides were separated and quantified by HPAEC-PAD on a Dionex

CarboPac PA1 column and precolumn under the following conditions: sodium
hydroxide/water gradient at 35 oC; flow rate 1 mL min-1. Postcolumn addition of 500 mM Commented [AW[29]: Should include concentrations
NaOH at a flow rate of 0.4 mL min-1 was used for detection (Gosselink et al., 2010). The
ash content was measured thermogravimetrically, using a Perkin-Elmer Pyris 1
thermogravimetric analyser, based on the method adapted from Hodgson et al., (2011).
Uronic acids were spectrophotometrically determined in the hydrolysates at wavelength
of 520 nm according to the method of Blumenkrantz and Asboe-Hansen, 1973. All of the
analyses were performed in duplicate on technical replicates.
3.3.2.2 Solubility
The solubility of lignin in organic solvents was assessed based on the method adapted
from Cybulska et al., (2012) using methanol, ethanol, ethyl acetate, dioxane and dimethyl
sulfoxide (DMSO). 50 mg of lignin fractions were weighed in to a 15 mL falcon tube and Commented [AW[30]: Size of tube?
2.5 mL of different organic solvents were added. The solutions were kept at 40 oC with
constant agitation for 48 hours. After that, solutions were filtered through filterpaper
(Whatman No.1) and the solid residue was dried at 70 oC. The percent solubility of the
lignin sample in a particular solvent was based on the comparison between initial sample
weight and remaining solid residue after incubation in solvent. All of the analyses were
performed on triplicate technical replicates.
3.3.2.3 31P- NMR

The presence of functional groups was determined by 31P-NMR. Lignin samples were
analysed according to the method described by Gosselink et al., (2010). 30 mg of lignin
fractions were added to a 1 mL vial and mixed with 100 µl DMF/pyridine (1:1 v/v) and
100 µl internal standard solution containing 15 mg mL-1 cyclohexanol (internal standard)
and 2.5 mg mL-1 chromium (III) acetylacetonate in pyridine. This suspension was stirred
for 16 hours at room temperature. Derivatization reagent (2-chloro-4,4,5,5-tetramethyl-
1,3,2-dioxaphopholane) (100 µL) was mixed with 400 µL of CDCl3 prior to addition to the
lignin suspension. After mixing, the mixture was analysed by NMR (Bruker 400 MHz),
with 30 oC pulse angle, inverse gated proton decoupling, a delay time of 5s and 256 scans.
102
The results were expressed on a dry lignin weight basis. All of the analyses were
performed on duplicate technical replicates.
3.3.2.4 FTIR
FTIR was performed on all lignin samples. Lignin spectra were collected by attenuated
total reflectance (ATR) in the range 4000-700 cm-1 using an Equinox 55 FTIR
spectrometer (Bruker Optik, Ettlingen, Germany), equipped with a Golden Gate ATR
accessory (Specac, Slough, UK). Spectra were collected over 32 scans at a resolution of 4
cm-1 and corrected for background absorbance. Full spectra were normalized to the
maximum absorption value. All of the analyses were performed on duplicate technical
replicates.
3.3.2.5 TGA
Thermogravimetric analysis (TGA) was used to determine the thermal stability,
decomposition temperature and ash content for each lignin fraction extracted from
different sources. TGA experiments were carried out using a Perkin-Elmer Pyris 1
thermogravimetric analyser, based on the method adapted from Hodgson et al., (2011).
TGA (weight loss as a function of temperature) and derivative thermogravimetry (DTG)
curves were recorded from room temperature to 905 oC. The temperature programme
which was used is described below: samples were held at 30 oC for 5 minutes, then
heated from 30 oC to 105 oC over 25 min, held at 105 oC for 5 minutes, heated from 105
oC to 905 oC over 25min, held at 905 oC for 15 minutes and cooled from 905 oC to 200 oC
over 200min. For the ash content, samples were heated from 200 oC to 575 oC over 25
min and held at 575 oC for 50 minutes and then for 1 minute. All of the analysis was
performed on triplicate technical replicates.
3.3.2.6 Molecular weight (SEC)

Molar mass distribution of lignin was determined using alkaline Size Exclusion
Chromatography (SEC) based on a method adapted from Gosselink et al., (2010). Lignin Commented [AW[31]: Is there any information about the hplc
system
samples of 1 mg were dissolved in 0.5 M NaOH and injected onto a column set consisting
of a TSK gel guard column PWxl (6 mm x 4 cm, particle size: 12µm) and two serial
connected columns TSKgel GMPWxl (7.8mm x 30 cm, particle size: 13 µm) and eluted
with the same solvent. Conditions were as follows: flow rate 1 mL min-1, column
temperature 25 oC, and detection at 280 nm. Standards for calibration of the molar mass
103
distribution were a series of sodium-polystyrene sulphonates (mw range 891 to 976,000
Dalton) and phenol. Mw (weight average molecular weight), Mn (number average
molecular weight) and polydispersity (PD, Mw/Mn) were calculated. All of the analysis
was performed in technical duplicate replicates.
3.4 EXPERIMENT I: LIGNIN CHARACTERISATION FROM GRASSES

3.4.1 RESULTS
3.4.1.1 Lignin composition
Lignin composition was analysed using protocols described in section 3.3.2.1. Table 3.1
shows the ash, sugar residue, Klason lignin (Acid Insoluble Lignin) and Acid Soluble Lignin
content (on a dry matter basis) of lignin fractions extracted from various feedstocks. The
results of the analyses showed, that source of lignin and biomass pre-treatment
influenced the content of Klason lignin and contaminants (ash and sugar residues). The
purity of all tested samples indicated by Klason lignin content ranged between 84.65 % - Commented [AW[32]: Round up values to one decimal place
90.06 %. Most of the lignin fractions showed <5 % content of carbohydrates and ash
impurities. The purity of lignin fractions extracted in this study was much higher
compared to the CIMV WS lignin fraction, which exhibited a Klason lignin content of
84.56 %. The highest Klason lignin content observed was 90.06 % in the case of WS SE
Acid lignin. This lignin fraction also contained a relatively small amount of carbohydrates
impurities compared to other lignin fractions (2.54 %). However, it contained an ash
content of 4.53 % which was the highest among all of the tested samples. CS lignin
showed the lowest purity with a Klason lignin content of 84.65 %. While the amount of
carbohydrates impurities in this lignin was quite high with a value of 4.18 %, the ash
content was very low (0.62 %). With respect to lignin fractions extracted from non- Commented [AS[33]: I left this bit for both wood and grass
analyses as I think it just mentions the lowest and the highest purity,
pretreated materials, the sugar residue content observed for WS, CS and MG, was: 4.80, not each individual sample, this is of high importance as based on
this mostly I pick up WS SE Acid as the lignin with the best properties
for polyurethanes, pf resins etc, which is one of the key findings. I
4.18 and 2.85 %, respectively. Steam explosion under acetic and alkaline conditions think its worth to mention it, otherwise my conclusions wont make
too much sense.
decreased the amount of sugar residues in lignin extracted from WS (2.54 % for SE Acid
and 2.02 % for SE Alk compared with 4.8 for WS) and CS (2.24 % for SE Acid and 2.16 %,
for SE Alk compared with 4.18 for CS) and the differences were mostly observed in case
of xylan and arabinan. The amount of uronic acid also dropped following steam explosion
pre-treatment. The only exception was reported with MG lignin fraction, which exhibited
a higher content of sugar residues, following steam explosion process under acetic
104
conditions compared with non-pretreated extracts (3.54 % vs 2.94 % respectively). It has
been observed, that the amount of glucan markedly increased in MG SE Acid lignin
fraction. Similar pattern was reported in case of uronic acid, exhibiting higher amount
following steam explosion under acetic conditions. In contrast, the amount of
carbohydrate impurities present in MG lignin extracted from steam exploded material
under alkaline conditions was much lower (2.50 %). All lignin samples extracted from
steam exploded material with alkaline treatment, showed a lower ash content compared
with other lignin fraction. In contrast, the ash content of lignin fractions extracted from
steam exploded material treated with acid treatment was higher. The highest and lowest
ash content exhibiting 4.53 % and 0.28 % was respectively observed in the case of WS SE
Acid lignin and MG SE Alk lignin. Commented [AW[34]: Not sure this observation is worth
making
105
Table 4.121: Analysis of lignin extracts from non-pretreated and acid or alkali steam exploded Miscanthus giganteus, wheat straw and corn stover. Values are presented as a
percentage of dry matter (DM). Standard deviation (SD) of the mean (n=2) is 0. ND= not detected.
Composition MG MG SE Acid MG SE Alk CS CS SE Acid CS SE Alk WS WS SE Acid WS SE Alk CIMV W.S

lignin lignin lignin lignin lignin lignin lignin lignin lignin lignin
Total Carbohydrate 2.94 3.54 2.50 4.18 2.24 2.16 4.80 2.54 2.02 3.02
composition
Arabinan 0.86 0.67 0.51 0.76 0.62 0.21 1.20 0.84 0.20 0.23
Xylan 1.11 0.71 0.84 0.91 0.43 0.41 1.19 0.68 0.51 0.67
Mannan 0.05 0.03 0.05 0.04 0.02 0.09 0.06 0.03 0.04 0.13
Galactan 0.07 0.03 0.07 0.27 0.19 0.10 0.09 0.04 0.07 0.15
Glucan 0.80 1.34 0.58 1.24 0.73 0.87 1.11 0.43 1.06 0.99
Rhamnan 0.02 0.01 0.01 0.03 0.01 0.03 0.03 0.01 0.01 0.03
Uronic acid 0.04 0.76 0.43 0.93 0.23 0.45 1.11 0.50 0.12 0.82
Total lignin 87.86 88.71 89.01 85.84 89.42 87.57 90.35 90.57 87.84 85.89 Commented [AW[35]: Round up these values to one decimal
Klason lignin 87.28 88.22 88.47 84.65 88.13 86.33 89.83 90.06 87.27 84.56 place
Acid Soluble Lignin 0.58 0.48 0.55 1.19 1.29 1.24 0.52 0.51 0.57 1.33
Ash 1.43 2.94 0.28 0.62 1.42 0.6 2.36 4.53 0.64 N/D
106
3.4.1.2 Chemical properties and structure
3.4.1.2.1 Solubility
Lignin solubility was tested as described in section 3.3.2.2. Table 3.2 shows the solubility
of lignin fractions in different organic solvents on dry matter basis. The results of the
analysis showed, that all lignin samples were generally soluble to some degree in tested
solvents. Very high solubility of lignin fractions with values >76 % was observed with
dioxane and DMSO. WS lignin fractions were more soluble than CS and MG lignin
fractions, with WS lignin exhibititng the highest solubility in dioxane (96.05 %). All lignin Commented [AW[36]: Round up values to one decimal place
fractions extracted from Miscanthus giganteus and corn stover exhibited >90 % solubility
Worth pointing out that for non-rpretreated lignin fractions WS
in DMSO. The highest solubility (97.16 %) was observed for MG SE Acid lignin. Ethanol, lignin was most soluble in most solvents
showed very limited effectiveness for for solubilizing all lignin fractions with values In the case of acid treated MG and CS – there is increase in sol-in
most solvents- no consistent pattern in case of alkali treated
ranging from 40.01 %-53.56 %. Similar results were observed for ethyl acetate, which Commented [AS[37]: I left it as I mentioned the highest value
for solubilityi in dioxane
exhibited lignin solubility ranging between 42.24 % - 57.15 %. With regard to pre-
treatments, MG and CS lignin fractions extracted from acid and alkali treated material
showed an obvious increase in solubility in most of the tested solvents. In turn, solubility
of WS lignin fractions extracted from non-pretreated feedtocks was higher comparing to
lignin fractions generated from acid treated material. Alkali pre-treatment did not show
an obvioius effect on WS lignin solubility in tested solvents.
107
Table 4.132: Solubility of lignin extracts in different organic solvents. Values are presented as a percentage of dry matter (DM) with standard deviation (SD ±) of the mean
(n=3).
Lignin type Methanol Ethanol Ethyl acetate Dioxane DMSO
MG 52.09 ± 1.143 40.01 ± 0.484 46.70 ± 0.260 88.35 ± 0.120 90.57 ± 0.101
MG SE Acid 61.88 ± 2.017 42.87 ± 0.439 50.62 ± 0.257 80.81 ± 1.009 97.16 ± 0.443
MG SE Alk 56.51 ± 1.172 40.46 ± 0.678 50.06 ± 0.112 92.19 ± 0.680 96.14 ± 0.112
CS 50.65 ± 0.627 45.74 ± 0.940 48.54 ± 0.449 86.63 ± 0.990 90.83 ± 0.104
CS SE Acid 59.0 3 ± 0.645 49.22 ± 1.183 46.04 ± 0.939 76.84 ± 0.346 92.96 ± 0.770
CS SE Alk 55.96 ± 0.765 53.56 ± 0.934 42.24 ± 0.874 76.41 ± 0.549 94.69 ± 0.549
WS 64.92 ± 0.926 46.15 ± 0.871 57.15 ± 0.242 96.05 ± 0.815 88.80 ± 0.076
WS SE Acid 60.94 ± 0.491 41.37 ± 0.231 54.94 ± 0.889 94.50 ± 0.235 87.85 ± 1.096
WS SE Alk 53.95 ± 0.428 50.62 ± 0.595 54.90 ± 0.541 95.11 ± 0.780 92.89 ± 0.657
108
3.4.1.2.2 31P-NMR
The presence of functional groups was determined by 31P- NMR as described in section
3.3.2.3. An example of a 31P-NMR spectrum of lignin extracted from CS SE Acid is shown
in Figure 3.1. Table 3.3 shows the amount of hydroxyl groups, consisting of aliphatic OH,
condensed phenolic OH, syringyl OH, guaiacyl OH, p-hydroxyl OH, and COOH groups. The
concentration of each hydroxyl functional groups (mmol g-1) was calculated on the basis
of the hydroxyl content of the internal standard and its peak area. The results of the
analysis showed, that MG lignin fractions had the highest amount of total OH groups with
the values ranging from 4.81-5.03 mmol g-1. In turn, CS lignin fractions exhibited the
lowest amount of total OH groups with the values ranging from 3.69-4.69 mmol g-1. The
total amount of OH groups for WS lignin fractions ranged from 4.63-4.73 mmol g-1 (Figure Commented [AS[38]: It is worth expanding on this- group
species- all miscanthus higher- CS generally lower/WS inbetween-
3.2). Aliphatic OH groups formed the largest component of total OH content with the Ms high G and S
CS- generally low etc
highest content observed for WS lignin, which exhibited a value of 2.14 mmol g-1 and the For standard lignins- add up values you have to give an estimate of
total
lowest amount of 1.13 mmol g-1 observed for CS SE Acid lignin. It has been observed,
that the amount of aliphatic groups was much lower in the case of lignin fractions Say in general miscanthus and wheat straw show symilar level of
hydroxyl groups witht he exception of P-hyroxul , take out the
extracted from steam exploded material. In turn, it is notable that levels of other OH highest and the lowest amount with each groupo
groups were generally higher in lignin fractions extracted from pre-treated material
(Figure 3.3). In general, MG and WS lignin fractions exhibited similar level of different OH
groups comparing to CS lignin fractions. The amount of aliphatic OH groups in MG and
WS lignin fractions with the values ranging from 1.75-2.02 and 1.54-2.14 mmol g-1,
respectively was much higher comparing to CS lignin fractions exhibiting values ranging
from 1.13-1.47 mmol g-1. A similar observation was made for syringyl OH and guaiacyl
OH groups with syringyl OH and guaiacyl OH values for MG lignin fractions ranging from
0.42-0.58 mmol g-1 and 0.72-0.86 mmol g-1 respectively, while respective values for WS
lignin fractions ranged from 0.40-0.55 mmol g-1 and 0.78-0.91 mmol g-1. This was much
higher comparing to CS lignin fractions of which the syringyl OH and guaiacyl OH content
respectively ranged from 0.26-0.41 mmol g-1 and 0.49-0.76 mmol g-1. Additionally, MG
and WS lignin fractions exhibited condensed phenolic OH content with the values
respectively ranging from 0.53-0.72 mmol g-1 and 0.42-0.69 mmol g-1. This was much
higher comparing to CS lignin fractions of which condensed phenolic OH content ranged
from 0.36-0.57 mmol g-1. The only exception was seen in case p-hydroxyl OH and COOH
groups. In case of WS lignin fractions, the amount of p-hydroxyl groups (0.33-0.43 mmol
109
g-1) was much lower comparing to MG and CS lignin fractions (0.66-0.70 mmol g-1 and
0.59-0.64 mmol g-1 respectively). In turn, CS lignin fractions exhibited much higher
content of COOH groups (0.52-1.08 mmol g-1) comparing to MG and WS lignin fractions
(0.41-0.62 mmol g-1 and 0.48-0.82 mmol g-1 respectively). All lignin samples showed
different results to the lignin standards. The amount of aliphatic, condensed phenolic,
and guaiacyl OH groups in lignin fractions tested in this study was lower comparing to
Kraft Indulin AT (Softwood) lignin. In contrast to Alcell lignin, the amount of syringyl OH Commented [AW[39]:
I don’t think you can say this, Kraft lignin is quite different
groups was also slightly lower. In turn, comparing to lignin analysed in this study, Kraft
Worth highlighting that values are very high in Kraft
and Alcell lignin standards exhibited markedly lower content of p-hydroxyl OH and COOH S very high in Alcell POH very low- so quite different
groups (Table 3.3).
110
Table 4.143: Contents of functional groups as determined by 31P- NMR. The range of OH groups include: aliphatic OH, cond.Phen.OH, syringyl OH, guaiacyl OH, p-Hydroxyl OH
and COOH groups (mmol g-1). Standard deviation (SD) of the mean (n=2) is 0. ND= not detected.
Aliphatic Condensed.
Syringyl OH Guaiacyl OH p-Hydroxyl OH COOH Total
Lignin type OH Phenolic
OH
MG 2.02 0.53 0.42 0.72 0.68 0.44 4.81

MG SE Acid 1.75 0.72 0.58 0.86 0.70 0.41 5.03
MG SE Alk 1.79 0.59 0.54 0.84 0.66 0.62 5.03
CS 1.47 0.36 0.26 0.49 0.60 0.52 3.69
CS SE Acid 1.13 0.45 0.32 0.59 0.59 0.63 3.72
CS SE Alk 1.24 0.57 0.41 0.76 0.64 1.08 4.69
WS 2.14 0.42 0.40 0.78 0.41 0.48 4.63
WS SE Acid 1.64 0.73 0.55 0.91 0.43 0.50 4.77
WS SE Alk 1.54 0.69 0.49 0.87 0.33 0.82 4.73
Alcell 1.08 0.76 1.05 0.70 0.20 0.30 4.09
Kraft Indulin AT (Softwood) 2.35 1.36 N/D 1.88 0.22 0.49 6.30
111
31
Figure 4.211: P-NMR spectra of CS SE Acid lignin.
112
Commented [AW[40]: Total content of functional groups
Total content of functional groups (mmol g-1)

Total functional OH groups (mmol g-1)
6
5
4
3
2
1
0
MG SE Acid MG MG SE Alk CS SE Acid CS CS SE Alk WS SE Acid WS WS SE Alk
Lignin
Figure 4.222:Total content of functional groups as determined by 31 P-NMR in mmol g-1. Standard deviation (SD) of the mean (n=2) is 0. Commented [AW[41]: Y axis title- could make this Total
functional OH – to match below and keep shorter
Legend – Total functional groups (mmol g-1)
113
Commented [AW[42]: I would use singular for lignin e.g. lignin
Functional group content not lignins
Commented [AW[43]: Fuctional group content – better title
2.5 Aliphatic OH
Cond.Phen OH
2
Functional OH groups (mmol g-1)
Syringyl OH
1.5
Guaiacyl OH
p-Hydroxyl
1
COOH
0.5
0
MG MG SE Acid MG SE Alk CS CS SE Acid CS SE Alk WS WS SE Acid WS SE Alk
Lignin
Figure 4.233: Contents of functional groups in different lignin fractions. Data represent aliphatic OH, cond.Phen.OH, syringyl OH, guaiacyl OH, p-Hydroxyl OH and COOH
groups in mmol g -1. Standard deviation (SD) of the mean (n=2) is 0.
114
3.4.1.2.3 FTIR
FTIR analysis was carried out following the method described in section 3.3.2.4. Table 3.4
gives the assignment of FTIR absorption bands of the lignin, based on the literature. As
seen in Figure 3.4 in all cases the spectral profiles were very similar, which confirmed the
‘core’ of lignin structure. The differences were mostly seen in the intensity of bands. All
lignin fractions showed the broad band at 3460-3410 cm-1 attributed to the hydroxyl
groups. In comparison with WS and MG, lignin extracted from CS demonstrated an
apparent higher intensity of aliphatic groups presented in the range of 2938-2842 cm-1. Commented [AW[44]: Not evident from figure 4.5 –WS also
looks high
Most peaks were observed between 1900-700 cm-1 and they are presented in Figure 3.5.
The bands with very similar intensity in all cases were observed in the range of 1707- Commented [AW[45]: Could say here- Most peaks were
observed between 1900-700 cm-1 and these are presented in Figure
1691 cm-1 indicating unconjugated aldehyde/ketone groups. The changes of the carbonyl 4.5.
absorption were observed at 1660 cm-1, where the signal indicating the bond, appeared
as a peak only in the case of lignin fractions extracted from CS and WS and not from MG.
This demonstrated, that WS and CS lignin fractions contained higher amounts of
conjugated carbonyl groups, compared with MG lignin, regardless of treatment.
Differences were also observed at 1167cm-1, indicating a much greater amount of ester
groups in the case of lignin extracted from CS and MG compared to WS lignin, regardless
of treatment. Aromatic skeleton vibrations in the ranges of 1615-1600 cm-1, 1515-1511 Commented [AW[46]: From figure 4.6 levels appear lowest in
WS lignin
cm-1 and 1425-1423 cm-1 were common for all lignin fractions with slight differences in
intensity. Also, the vibrations in the range of 1460-1459 cm-1, indicating C-H bending of
methyl and methylene groups were quite strong and common for all tested lignin
fractions. The spectra for all lignin samples showed characteristic vibrations for guaiacyl
unit (1267 cm-1) and weak vibrations for syringyl unit (1326 cm-1). Common for all the
lignin fractions were also weak vibrations at 1367 cm-1 (originating from aliphatic C-H in
methyl groups) and at 911 cm -1 (originating from C-H bending of syringyl units). In turn,
strong vibrations were seen in the range of 1219-1211 cm -1, (characteristic for C-C C-O
and C=0 stretching), 1123-1110 cm-1 (indicating aromatic C-H deformation of syringyl
units), 1031-1015 cm-1 (specific for C-0 stretching of primary alcohols) and 835-815 cm-
1 (indicating C-H bending of syringyl+hydroxyl units). The band at 983 cm-1 originating
from CH=CH groups, provides evidence for conjugative units particularly in the case of
lignin fractions extracted from CS and MG (a weak signal was observed for WS lignin in
115
this area) (Figure 3.4). As seen in Figure 3.5, a comparison of lignin fractions extracted
from pre-treated and non-pretreated materials showed, that the bands of the fingerprint
regions were very similar. The differences were mostly seen in the intensity of bands.
The detailed analysis of treatment and their influence on the chemical structure of lignin
is described below.
MG lignin
For lignin extracted from MG, the highest peak intensity was observed in the range of
1123-1110 cm-1 related to the aromatic C-H deformation of syringyl units. Differences
were only observed for a few regions. High intensity peaks were seen in the ranges of
1707-1691cm-1 (related to the unconjugated carbonyl stretching of aldehyde/ketone
groups), 1167cm -1 (related to the C-O stretching of ester groups), 1031-1015cm-1
(related to the C-O stretching of primary alcohols) and at 835-815 cm-1 (related to the C-
H bending of syringyl+hydroxyl units) for MG lignin and MG SE Acid lignin. In summary,
the chemical structure of MG SE Alk lignin was clearly distinguishable from the other two
fractions, however the differences were also observed only in the intensity of peaks
(Figure 3.6).
WS lignin
In the case of WS, all lignin fractions showed the highest peak intensity, with no
difference between treatments, in the range of 1123-1110 cm-1 related to the aromatic
C-H deformation of syringyl units. In addition, all lignin fractions showed very similar
intensity of peak in the range of 1707-1691 cm-1 (related to the unconjugated carbonyl
stretching of aldehyde/ketone groups) and at 1367 cm-1 (related to the aliphatic C-H
stretching in CH3). The similarity in peaks could be also observed between all lignin
fractions, for example: in the range of 1219-1211 cm-1 (related to the C-C, C-O and C=0
stretching of guaiacyl unit), at 1167 cm-1 (related to the C-O stretching of ester groups)
and at 1267 cm-1 (related to the C=O stretching of guaiacyl unit). The differences were Commented [AW[47]: Look again- I am not convinced that
they are that different to alk
only observed for a few regions. C-O stretching of primary alcohols in the range of 1031-
1015 cm-1 was particularly intense for WS lignin and high intensity of C-H bending of
syringyl+hydroxyl units in the range of 835-815 cm-1 was observed for WS SE Acid lignin.
116
In general, WS SE Alk lignin showed more intense peaks compared to WS lignin and WS
SE Acid lignin for bands above 1400 cm-1 (Figure 3.7).
CS lignin
All CS lignin fractions presented the highest intensity peak in the range of 1123-1110 cm-
1, related to the aromatic C-H deformation of syringyl units. In addition, all lignin
fractions showed very similar peak intensity in the range of 1707-1691 cm-1 (related to
the unconjugated carbonyl stretching of aldehyde/ketone groups), 1615-1600 cm-1
(related to C=C stretching of aromatic rings in lignin) and at 911 cm-1 (related to C-H
stretching of syringyl units, aromatic rings). However, C-O stretching of primary alcohols
in the range of 1031-1015 cm-1 was particularly intense for CS lignin. Similarly, a high
intensity of C-H bending of syringyl+hydroxyl units in the range of 835-815 cm-1 was
observed in CS lignin and CS SE Acid lignin. In the region of 1300-1600 general CS SE Alk
lignin fractions showed more intense peaks compared to CS SE Acid lignin and CS lignin
(Figure 3.8).
Comparison of lignin standards (CIMV and Kraft), with generated lignin fractions (using
MG SE Acid lignin as an example) showed, that the MG lignin produced a very similar
profile to CIMV lignin. The differences were mostly seen in the intensity of the peaks.
The band at 1167 cm-1 indicating C-O stretching of ester groups showed a higher intensity
in the case of MG SE Acid lignin. Also, the intensity of the peak in the range 835-815 cm-
1, indicating C-H bending of syringyl+hydroxyl units, was much higher in the case of MG
SE Acid lignin. The peak at 1660 cm-1 (indicating C=O conjugated stretching), showed a
lower intensity in the case of MG SE Acid lignin compared with CIMV lignin. The Kraft
lignin profile was distinctly different to that of MG SE Acid lignin and CIMV lignin. In the
case of MG SE Acid and CIMV lignin, characteristic peaks at 983 cm-1 (originating from
CH=CH bending) and in the range of 1425-1423 cm-1 (indicating C-H deformation in
lignin) were observed. The bands in the range of 1707-1691cm-1 (indicating
unconjugated carbonyl stretching of aldehyde/ketone groups) and at 1367 cm-1
(indicating aliphatic C-H stretching in CH3) were also noticed. In addition, in the case of
MG SE Acid lignin the typical band in the range of 835-815 cm -1 indicating C-H bending
of syringyl+hydroxyl units (typical for herbaceous lignin), 1326 cm-1 indicating C=O
stretching of syringyl groups and at 1167 cm-1 indicating C-O stretching of ester groups
117
was observed. In contrast, Kraft lignin showed a characteristic peak at 1085 cm-1,
(indicating C-O deformation in secondary alcohols and aliphatic ethers) and at 854 cm -1
(indicating C-H out of plane in position 2,5 and 6 of G units) which was not observed in
MG SE Acid and CIMV lignin samples. Other differences were mostly seen in the intensity Commented [AS[48]:
of bands. MG SE Acid and CIMV lignin showed much greater intensity in the range of Stick to one S+G or syringyl and guiacyl
1515-1511 cm-1, 1460-1459 cm-1, and 1425-1423cm -1 (Figure 3.9).
S/G ratio
S/G ratio has been calculated using normalized FTIR spectra with intensity of peaks at
1327 and 1268 cm-1 according to the method of Sammons et al., (2013). Table 3.5 shows
the results of S/G ratio in different lignin fractions. The results of the analysis showed, Commented [AW[49]: I would add something along lines – S/G
ratios lie within a narrow range (0.61-0.71)
that S/G ratios for all pre-treated and non-pretreated lignin fractions lie within a narrow
range (0.61-0.71). In the case of lignin fractions extracted from WS and CS, the lowest
S/G ratios were observed in non-pretreated samples while the highest were observed in
lignin extracted from SE Alk material. With regard to MG, the S/G ratio was the same for
all tested samples (Table 3.5).
118
Table 4.154: Peak assignments for the FTIR spectra (Boeriu et al., 2004, El Hage et al., 2009, Kline et al.,
2010, Li et al., 2009, Monteil-Rivera et al., 2013).
Observed peaks (cm-1 ) Peak assigment
3460-3410 O-H stretching

2938-2842 Aliphatic
1707-1691 Unconjugated carbonyl stretching of
aldehyde/ketone groups
1660 C=O stretching conjugated
1615-1600 C=C stretching of aromatic ring in lignin
1567 C=O stretching (ionic compounds only)
1515-1511 C=C stretching of aromatic ring in lignin
1460-1459 C-H bending of methyl and methylene groups
1425-1423 C-H deformation in lignin
1367 Aliphatic C-H stretching in CH3
1326 C=O stretching of the syringyl unit
1267 C=O stretching of guaiacyl unit
1219-1211 C-C, C-O and C=O stretching of guaiacyl unit
1167 C-O stretching of ester groups
1123-1110 Aromatic C-H deformation of syringyl units
1085 C-O deformation in secondary alcohols and aliphatic
ethers
1031-1015 C-O stretching of primary alcohols
983 CH=CH bending
911 C-H bending of syringyl units, aromatic rings
899 C-H deformation vibration of cellulose
854 C-H out of plane in position 2,5 and 6 of G units
835-815 C-H bending of S+H units
119
Table 4.165: S/G ratio based on FTIR method.
Lignin sample S/G ratiio (FTIR)
WS SE Acid 0.65
WS 0.61
WS SE Alk 0.69
CS SE Acid 0.67
CS 0.64
CS SE Alk 0.71
MG SE Acid 0.62
MG 0.62
MG SE Alk 0.62
120
Figure 4.244: FTIR spectra of all extracted lignin fractions.
121
A
Figure 4.255: FTIR spectra of lignin fractions extracted from pre-treated (SE Acid, SE Alk) and non-pretreated material: A; WS, B; MG, C:CS.
122
Figure 4.266: FTIR spectra of MG lignin fractions extracted from steam exploded (SE Acid, SE Alk) and non-pretreated materials.
123
Figure 4.277: FTIR spectra of WS lignin fractions extracted from steam exploded (SE Acid, SE Alk) and non-pretreated materials.
124
Figure 4.288: FTIR spectra of CS lignin fractions extracted from steam exploded (SE Acid, SE Alk) and non-pretreated materials.
125
Figure 4.299: FTIR spectra of MG SE Acid lignin (example) and lignin standards (CIMV, Kraft lignin).
126
3.4.1.2.4 TGA
Thermal stability ,decomposition temperature and ash content of all lignin samples were
determined by TGA according to the protocol presented in section 3.3.2.5. TG curves
revealed the weight loss of substances in relation to the temperature of thermal
degradation, while the first derivative of the curve (DTG) showed the corresponding rate
of weight loss. All lignin samples revealed 63.10 % - 67.78 % DM volatile content and
29.83 % - 33.82 % DM fixed carbon content. The ash content ranged between 0.28 % -
4.53 % DM (Table 3.6). As can be seen from the TGA/DTG curves, all lignin fractions
showed very similar trends (Figures 3.10 and 3.11). The initial weight loss, was due to
water evaporation (Wang et al., 2014). From 130 oC to 450 oC, the degradation was due
to the breakage of C-C linkages between the phenolic units causing structural
rearrangements and evaporation of phenolic compounds (Wörmeyer et al., 2011). The
peak temperature (Tmax) corresponding to the maximum decomposition rate, occurred
in the range of 384 -404 oC, with the lowest value observed for CS SE Acid lignin and the
highest for WS lignin (Table 3.6). Pyrolytic degradation in this region involved
fragmentation of inter-unit linkages releasing monomeric phenols into the vapour phase
and above 500 oC the process was related to the decomposition of some aromatic rings
(Tejado et al., 2007). The weight loss of lignin fractions slowed at temperatures >500 oC.
At temperatures >700 oC the weight loss of the lignin fractions reached a plateau (Figures
3.10 and 3.11). The main differences between all degradation curves appeared in the
range of 200 -300 oC (Figure 3.11). A specific shoulder, was noted in MG and WS lignin
fractions extracted from SE Acid treated material and in MG lignin extracted from SE Alk
material. The DTG profile of CS lignin was very similar, regardless of treatment (Figures
3.13 and 3.14). Therefore, the weight loss profile depended both on lignin species source
and pre-treatment. As can be seen in Figure 3.15 presenting second derivative (DTG)
curves, which emphasize differences in rates, the difference between lignin fractions
generated from steam exploded and non-pretreated materials in the range of 200 -300
oC were substantial.
127
Table 4.176: Proximate analysis of lignin samples determined from TGA graphs. Volatiles, fixed carbon
and ash are represented as a percentage of dry matter (DM). Standard deviation of the mean (n=3) is 0.
Lignin type Volatiles Fixed Carbon Ash Tmax
WS SE Acid 64.79 30.61 4.53 403.48

WS 63.10 30.68 2.36 403.58
WS SE Alk 65.73 30.17 0.64 393.58
CS SE Acid 66.31 32.13 1.42 383.53
CS 67.78 29.83 0.62 386.24
CS SE Alk 64.91 32.07 0.60 378.69
MG SE Acid 63.73 30.21 2.94 399.47
MG 64.40 33.51 1.43 387.81
MG SE Alk 62.92 33.82 0.28 397.04
128
120
100
WS SE Alk lignin
80 CS SE Alk lignin
Mass loss (%)
MG SE Alk lignin
60 WS lignin
CS lignin
MG lignin
40
WS SE Acid lignin
CS SE Acid lignin
20
MG SE Acid lignin
0
0 100 200 300 400 500 600 700 800 900 1000
Temp oC
Figure 4.3010: Thermogravimetric (TG) curves of WS, CS and MG lignin fractions extracted from steam exploded (SE Acid, SE Alk) and non-pretreated material. Commented [AW[50]: try and keep coloiur codes consistent in
figures below- also make sure colours can be easily seen
129
10
8
WS SE Alk lignin
Derivative weight (%/0 C)
7
CS SE Alk lignin
6 MG SE Alk
5 WS lignin
CS lignin
4
MG lignin
3 WS SE Acid lignin
2 CS SE Acid lignin
MG SE Acid lignin
1
0
90 190 290 390 490 590 690 790 890
Temp oC
Figure 4.3111: Differential thermogravimetric (DTG) curves of WS, CS and MG lignin fractions extracted from steam exploded (SE Acid, SE Alk) and non-pretreated material.
130
100 10
90 WS lignin 9
80 8
70 CS lignin 7
Mass loss (%)
60 6
MG lignin
50 5
40 4
30 3
20 2
10 1
0 0
0 200 400 600 800 1000
Temp °C
Figure 4.3212: Thermogravimetric (TG) and Differential thermogravimetric (DTG) curves of WS, CS and MG lignin samples fractions from non-pretreated material.
131
10
100
9
90 WS SE Acid lignin
8
80
7
70 CS SE Acid lignin
Mass loss (%)
60 6
50 5
MG SE Acid lignin
40 4
30 3
20 2
10 1
0 0
0 200 400 600 800 1000
Temp °C
Figure 4.3313: Thermogravimetric (TG) and Differential thermogravimetric (DTG) curves of WS, CS and MG lignin fractions extracted from steam exploded material with acid
treatment (SE Acid).
132
100 10
90 9
80 WS SE Alk lignin 8
70 7
CS SE Alk lignin
Mass loss (%)
60 6
50 MG SE Alk lignin 5
40 4
30 3
20 2
10 1
0 0
0 200 400 600 800 1000
Temp °C
Figure 4.3414: Thermogravimetric (TG) and Differential thermogravimetric (DTG) curves of WS, CS and MG lignin fractions extracted from SE material with alkaline treatment
(SE Alk).
133
A
Figure 4.3515: Second derivative DTG curves of lignin fractions from different materials (pre-treated
with steam explosion and non-pretreated): A; non-pretreated, B; SE Acid, C; SE Alk.
134
3.4.1.2.5 Molecular weight distribution (SEC)
The molecular weight distribution of lignin samples were obtained by Size Exclusion
Chromatography (SEC), following the method presented in section 3.3.2.6. Number-
average (Mn), Weight- average (Mw) molecular weights and polydispersity (Mw/Mn) of
the all lignin samples are shown in Table 3.7. As can be seen below, the Mm, Mw and
Mw/Mn ranged from 569-762 Dalton, 3018-7660 Dalton and 5.1-10.1, respectively.
Most of the lignin fractions extracted from steam exploded material with acid and
alkaline treatment showed higher molecular weights. The only exception was seen in the
case of WS SE Acid lignin, which showed a lower molecular weight compared to WS and
WS SE Alk lignin. The highest values of average weight molecular masses were observed
for CS SE Alk lignin (Mn: 762 Dalton, Mw: 7660 Dalton) and the lowest for WS SE Acid
lignin (Mn:588 Dalton, Mw: 3018 Dalton) and WS SE Alk lignin (Mn:569 Dalton, Mw:
3602Dalton). In general, the average molecular number (Mn) was higher for CS lignin Commented [AW[51]: I think you should point ojut that the
average molecular number was generally higher for CS lignin
fractions and lower for WS lignin fractions. Most of the lignin fractions showed a fractions and lower for WS fractions
Polydispersity were in similar range rather than higher and teh lower
comparable degree of polydispersity (Mw/Mn: 5.1-6.9), which was comparable with
Alcell lignin (Mw/Mn: 4.3), with the exception of CS SE Alk lignin (Mw/Mn: 10.1). In
addition, with the exception of CS SE Alk lignin, all lignin samples showed lower
polydispersity than CIMV lignin (Table 3.7). The molecular weight distribution curves for
WS lignin fractions were unimodal while the molecular weight distribution curves of for
MG and CS lignin fractions were bimodal. Differences between peaks were observed for
lignin fractions extracted from SE Acid, SE Alk and non-pretreated materials. (Figure
3.16). The molecular weight distribution of WS lignin fractions was very similar to CIMV
lignin (also a wheat straw derived lignin), showing unimodal curve (Figure 3.17) while the Commented [AW[52]: I don’t think you can say this- much
braoder distribution with CIMV lignin, all you can say is both show
molecular distribution of MG lignin fractions (Figure 3.18) and CS lignin fractions (Figure unimodal curve
3.19) was bimodal, showing the presence of a distinct low-molecular weight fraction.
Similarly to Alcell lignin, CS and MG lignin fractions, showed the presence of low-
molecular weight fractions. (Figures 3.17, 3.18 and 3.19).
135
Table 4.187: Average molar mass data (Mw, Mn) and polydispersity (Mw/Mn) of all lignin samples.
Standard deviation of the mean (n=2) is 0.
Lignin type Avg Mn (Dalton) Avg Mw (Dalton) Avg Polydispersity (Mw/Mn)
CS SE Acid 702 4824 6.9

CS SE Alk 762 7660 10.1
CS 710 3639 5.1
MG SE Acid 693 4019 5.8
MG SE Alk 671 4283 6.4
MG 693 3865 5.6
WS SE Acid 588 3018 5.1
WS SE Alk 569 3602 6.3
WS 593 3222 5.4
CIMV 907 8602 9.5
Alcell 681 2948 4.3
Lignin type SEC elution profiles
CS SE Acid
CS SE Alk
CS
MG SE Acid
136
MG SE Alk
MG
WS SE Acid
WS SE Alk
WS
CIMV
Alcell
Figure 4.3616: Size Exclusion Chromatography (SEC) profiles of grass lignin.
137
Figure 4.3717: SEC profiles of WS SE Alk, WS SE Acid and WS lignin fractions compared to lignin standards.
138
Figure 4.3818: SEC profiles of MG SE Alk, MG SE Acid and MG lignin fractions comparing to lignin standards.
139
Figure 4.3919: SEC profiles of CS SE Alk, CS SE Acid and CS lignin fractions compared to lignin standards.
140
3.4.2 DISCUSSION
Lignin offers potential for several applications in the field of biomaterials (Belgacem and
Gandini, 2008; Lora, 2008; Stewart, 2008; Doherty et al., 2011). They can be used as
green components in thermoplastics, or as a reagent in polyurethanes, polyesters, epoxy
or phenolic resin production (Monteil-Rivera et al., 2013). However, lignin’s structural
complexity and its dependence on feedstock and isolation process require
comprehensive analysis in order to assess, which material application is most suited for
a particular lignin (Constant et al., 2016).
General composition analysis typically includes Klason lignin, or Acid Insoluble Lignin
(AIL), Acid Soluble Lignin (ASL), residual carbohydrates and ash. In this study, all lignin
samples showed relatively high purity, containing over 87 % total lignin and 84 % of
Klason lignin and <5 % of carbohydrates and ash impurities (Xu et al., 2006). The purity
of lignin extracted from non-pretreated WS (89.83 % Klason lignin) was higher that of
CIMV wheat straw derived lignin standard (84.56 Klason lignin) (Table 3.1). In turn, results
presented by (Snelders et al., 2014) revealed that WS lignin with a purity of only 77.1 % Commented [AW[53]: should add material source of llignin
was obtained from a formic acid/acetic acid-based organosolv process. A high content
of carbohydrate residues in lignin extracted from non-pretreated WS and CS (4.80 % and
4.18 % respectively) indicated a strong association of hemicellulose and lignin in these
grass species and the occurrence of covalent bonds between lignin, hemicellulose and
cellulose has been previously reported in the literature (Monteil-Rivera et al., 2013).
According to Huijgen et al., (2014), carbohydrate impurities could also partly originate
from residual organosolv liquor, adhering to the precipitated and sedimented lignin. The
amount of sugar residues in WS lignin was slightly higher compared to CIMV wheat straw
derived lignin standard and slightly lower than that reported for CIMV wheat straw
derived lignin in a study by Snelders et al., (2014) with values of 3.02 % and 5.1 %
respectively. However, the feedstock source and conditions used for lignin extraction
were different, which could have affected the final composition. It is interesting, that
steam explosion with acid and alkaline treatment increased Klason lignin content in all
lignin fractions investigated in this study. The highest purity, exhibiting 90.06 % of Klason
lignin was seen in the case of WS SE Acid lignin. This lignin fraction also showed a lower
141
amount of carbohydrate impurities compared to lignin extracted from non-pretreated
feedstock (Table 3.1). As seen from results for other lignin fractions, the amount of sugar
residues decreased following steam explosion with acid and alkaline treatment. The
differences were mostly seen in case of xylan and arabinan which markedly dropped
after pre-treatment. This is most likely because most of the sugars have already been
removed during steam explosion (unpublished data), possibly via hydrolysis of ester
linkages. It’s also worth mentioning, that lignin fractions extracted from pre-treated
materials exhibited lower content of uronic acid, indicating removal of pectins. The only
exception was seen in the case of MG SE Acid lignin, which showed a higher amount of
carbohydrate impurities, especially glucan compared to lignin extracted from non-
pretreated MG (Table 3.1). This suggests that the increased content of carbohydrates
residues in pre-treated lignin was an effect of solubilized hemicellulose components
which were not removed during steam explosion becoming attached to lignin. Moreover,
increased amount of uronic acid, also suggested, that pectins have not been removed
during steam explosion and got attached to the lignin. However, detailed analysis of the
hydrolysate fraction and so called ‘pseudolignin’ (a term used to describe non-lignin
components in lignin fractions), would need to be carried out, in order to fully confirm
this theory. All lignin samples extracted from non-pretreated materials showed the ash
content ranging from 0.62 % to 2.36 % (Table 3.1). Samples with higher contents are in
agreement with data presented by Pan and Sano, 2005, showing that ash content in
herbaceous plants is much higher than that of wood (<1 %). Among all the samples, the
highest and lowest ash content with values of 4.53 % and 0.28 % were observed in the
case of WS SE Acid lignin and MG SE Alk lignin respectively. It is interesting, that steam
explosion under alkaline treatment markedly decreased the amount of ash in all studied
feedstocks (Table 3.1). This could possibly be due to the ash content being removed
during the pre-treatment. Han et al., (2010), have already reported decreased ash and
silica content after steam explosion. It has been suggested, that alkali treatment
depending on concentration and treatment time may either decrease or increase the
ash content. However, relatively mild alkali treatment for short period results in ash
removal (Mukherjee and Borthakur, 2004). A possible explanation is that alkali
conditions are effective for solubilising protein components which contain mineral
constituents (e,g, sulphur, iron, magnesium, copper). In turn, the amount of ash was
142
much higher in lignin fractions extracted from steam exploded material treated with acid
(Table 3.1). Generally speaking, loss of organic volatiles at harsh conditions (such as acid,
higher temperature, longer residence time) is the main reason causing the increase of
the ash (Horn et al., 2011). In addition, acidic conditions promote precipitation of protein
components.
Analysis of the solubility of lignin fractions in different organic solvents is also very
important, because it is known, that lignin has limited solubility in organic solvents,
mostly due to the heterogeneous three-dimensional structure (El-Khaldi Hansen et al.,
2016). The result of the comprehensive analysis carried out in this study showed, that
dioxane and DMSO were the best solvents exhibiting respectively >76 % and >90 %
solubility for all tested lignin samples (Table 3.2). Based on the Hildebrand theory, it was
predicted that when the solubility parameters of lignin and the solvent are the same, the Commented [AW[54]: what are solubility parameters?
Mention specific characteristics that make DMSO goood not sure
ability to dissolve the lignin will increase. Therefore, DMSO with a similar solubility about this
depends on aliphatic groups for interacting with polar

parameter as lignin was proposed to be one of the best solvents for lignin (Sameni et al.,
2017). Results presented in this study, were in close accordance with Cybulska et al.,
(2012) who demonstrated very high solubility of organosolv lignin samples in dioxane.
Higher or lower solubility of lignin fractions obtained from steam exploded material
(both with acid and alkaline treatment) could be due to the presence or absence of Commented [AW[55]:
impurities or different molecular weights. Generally speaking, lignin with higher In the case of acid treated MG and CS – there is increase in sol-in
most solvents
molecular weight reveals lower solubility in organic solvents (Sameni et al., 2017). This
pattern has already been seen in previous studies on lignin (Tables 3.2 and 3.7). As seen Commented [AW[56]: Also worth pointing out that Sameni
states that solubility also related to size and number of aliphatic
in this study, MG and CS lignin fractions extracted from acid and alkali treated material hydroxyl groups- worth looking at your data to see if any relationship
showed an obvious increase in solubility in most of the tested solvents. This was
consistent with lignin composition results, which revealed much lower amount of sugar
impurities in these fractions (Tables 3.1 and 3.2). Sameni et al., (2017) also states, that
lignin solubility can relate to number of aliphatic hydroxyl groups. The results presented
in this study were in close accordance with outcomes presented by this author. Higher
solubility was observed for lignin fractions generated from pre-treated MG and CS,
revealing lower amount of aliphatic groups comparing to lignin fractions extracted from
non-pretreated feedstocks (Tables 3.3 and 3.7). Altough, different patterns were likely to
occur, it was suggested, that the ability of a solvent to dissolve a variety of isolated lignin
143
fractions can be increased by increasing the hydrogen-binding capacity of the solvent.
This is because the hydrogen bonding is a major molecular interaction between hydroxyl
groups of lignin and organic solvents (Horvarth, 2005).
31P-NMR spectroscopy technique has been established to be a very powerful tool for
the characterization of phenolic hydroxyl (p-hydroxyphenyl, guaiacyl, and syringyl
structures), aliphatic hydroxyl and carboxylic acid groups present in the lignin samples
(Cateto et al., 2008). Results of the analysis carried out in this study showed, that
aliphatic OH groups showed the highest content of the total OH groups observed
(highest observed value of 2.14 mmol g-1 in WS lignin) (Table 3.3/Figure 3.3). It has been
proposed, that acetic and formic acid treatments tend to liberate β-O-4 bonds and
resulting in lignin fractions with high levels of OH aliphatic groups (Villaverde et al., 2010).
It is interesting, that the amount of aliphatic groups markedly decreased and the amount
of condensed phenolic groups increased, following steam explosion pre-treatment
(Table 3.3). Similar findings have previously been reported by Maniet et al., (2017). A Commented [AW[57]: Need to note that it could be due to
acid/alkali treatment- alkali treatment will favour condensation
reduction in the amount of aliphatic groups could be linked to do decrease in sugar reactions
Worth commenting that COOH high in alkaline treated samples (NB

residues which was evident in pre-treated lignin fractions (Table 3.3) (Huijgen et al., – oxidising conditions can cause oxidation of OH to COOH)
2014). The higher content of COOH groups in most of the lignin samples extracted from
pre-treated steam exploded material possible relates to oxidation (Sun et al., 2014). This
was most notable in MG, WS and CS alkali treated samples which would been subject to
strong oxidising conditions and will favour condensation reactions. It’s well known that
oxidising conditions can cause oxidation of OH to COOH. All lignin samples showed Commented [AS[58]: I think these are big differences for the
kraft lignin. I would say it is quite different. Mention G units are high
different results to the lignin standards. The amount of aliphatic, condensed phenolic, in softwood – so high G fits
Alcelll also quite different- if alcell comes from hard wood this wood
account for high S
and guaiacyl OH groups in lignin fractions tested in this study was lower comparing to
Kraft Indulin AT (Softwood) lignin. In contrast to Alcell lignin, the amount of syringyl OH
groups was also slightly lower. In turn, comparing to lignin analysed in this study, Kraft
and Alcell lignin standards exhibited markedly lower content of p-hydroxyl OH and COOH
groups (Table 3.3). However, this was probably caused by the different extraction
method and lignin source (wood). In general softwood lignin such as Kraft Indulin AT
reveal high amount of guaiacyl OH groups, typical for this type of feedstock. In turn, the
presence of syringyl OH groups is typical for hardwood lignin such as Alcell lignin (Jingjing,
2011).
144
The FT-IR spectra for all organosolv lignin fractions were very similar, confirming that the
"core" of lignin structure does not change dramatically during the various treatments.
Based on presented spectra, all lignin fractions were typical herbaceous lignin with SGH
units (Figure 3.4). As seen in Figure 3.5, a comparison of lignin fractions extracted from
pre-treated and non-pretreated materials showed that the bands of the fingerprint
regions were very similar. This observation highlighted the fact, that steam explosion
pre-treatment did not significantly change the core of lignin. However, it’s worth to add, Commented [AS[59]:
that peak observed in the range of 1031-1015 cm-1 (specific for C-0 stretching of primary 1031-1015 cm Nb- this peak is low for all alk treatments – need to
point this out mention in discuussuon that after alkalien teratment
this band is lower (oxidation)
alcohols) showed lower intensity for all lignin fractions extracted from alkali treated
material. This was most likely because of the strong oxidation conditions observed during
this type of pre-treatment. Compared to lignin standards (CIMV and Kraft), lignin
generated from combined steam explosion-organosolv process showed very similar
structure to CIMV lignin. The differences were mostly seen in the intensity of the bands.
In contrast, lignin samples showed marked difference when compared to Kraft lignin.
The presence of bands in the range of 1707-1691 cm-1 (indicating unconjugated carbonyl
stretching of aldehyde/ketone groups) has been shown to be characteristic for
organosolv lignin, and originate from carbonyl groups released during acid hydrolysis
(which may explain why it is not observed in the case of Kraft lignin) (Tejado et al., 2007).
The band at 1085 cm-1, (indicating C-O deformation in secondary alcohols and aliphatic
ethers) and at 854 cm -1 (indicating C-H out of plane in position 2,5 and 6 of G units,
typical for Kraft lignin (Li et al., 2009) has not been observed in organosolv lignin (Figure
3.9). All lignin fractions extracted from non-pretreated WS, MG and CS revealed the S/G
ratios of 0.61, 0.62 and 0.64 respectively (Table 3.5). These results were in close
agreement with data presented in the literature. An S/G ratio of 0.55 in WS lignin
obtained via the organosolv process has been reported by Huijgen et al., (2014).
Villaverde et al., (2010) reported a similar S/G ratio in organosolv lignin from MG, with a
value of 0.7. In turn, S/G ratio in organosolv lignin from CS, showing a ratio of 0.48 was
also observed previously by Sammons et al., (2013). It is interesting, that lignin fractions
extracted from steam exploded WS and CS showed increased S/G ratios compared to
lignin fractions obtained from non-pretreated materials (Table 3.5). These findings are in
contrast to findings from earlier studies carried out by Auxenfans et al., (2017), showing
lower S/G ratio in lignin extracted from pre-treated grass feedstocks. However, Wang et
145
al., (2017) observed, that increased S/G ratio might be a consequence of the prior
degradation of low molecular weight of non-condensed G-type lignin at relatively low
temperatures (184 oC, equivalent to 10 bars as used in this study). In this study research,
S/G ratio gradually declined as the temperature increased to 210 oC (equivalent to 15
bars). Commented [AW[60]: Good point
TGA was used to determine thermal stability of lignin fractions investigated in this study.
As can be seen from the TGA/DTG curves, all lignin fractions presented very similar
trends (Figures 3.10 and Figure 3.11). The main differences between all degradation
curves appeared in the range of 200 oC -300 oC (Figure 3.11). The specific shoulder seen
in MG and WS lignin fractions extracted from SE Acid (Figure 3.13) and in MG lignin
extracted from SE Alk material (Figure 3.14) showed the possible existence of a thermally
less stable domain within the lignin polymer (Toledano et al., 2011). That could
potentially indicate the degradation of a polysaccharide component. Hemicellulose, is
structurally amorphous, heterogeneous in composition and may contain different
combination of arabinose, galactose, mannose, xylose and decomposes mainly between
220 oC - 315 oC (Liu et al., 2014). The thermolysis reactions of polysaccharides occur Commented [AW[61]: PAPER BELOW IS WORTH A LOOK
BECAUSE IT BREAKS DOWN PARTS OF CURVE INTO DIFFERENT
COMPONENTS
through dehydration, decarboxylation, decarbonylation and the cleavage of glucoside
THERMOGRAVIMETRIC ANALYSIS AND KINETIC STUDY OF BAMBOO
bonds, C-H, C-O and C-C bonds (Sahoo et al., 2011). The presence of shoulder, indicating WASTE TREATED BY ECHINODONTIUM TAXODII USING A MODIFIED
THREE-PARALLEL-REACTIONS MODEL
polysaccharide components was already observed by Yu et al., (2015). It is worth Chen et al. Bioresource technol-
mentioning, that the presence of shoulders could also be caused by changes in the
chemical composition of lignin occurring in the process of steam explosion. During steam
explosion with acid treatment, some fractions of the hemicellulose and cellulose are
hydrolysed. After the condensation and recondensation reactions, combining lignin and
other degradation products might create a so-called ‘pseudolignin’. This phenomenon
has been previously observed with feedstocks pre-treated with acids (Sannigrahi et al.,
2011). However, according to recent studies, ‘pseudolignin’ was also observed in
feedstocks which were steam exploded with alkaline treatment. Based on these
observations, it’s very likely, that significant irregular peaks at temperatures between
200 oC -300 oC could be a signal of ‘pseudolignin’.
The number average molecular mass (Mn), weight average molecular mass (Mw) and
polydispersity (PD) of the lignin macromolecule, were determined by SEC. The results of
146
the analysis showed, that the highest values of average molecular mass was determined
in case of CS SE Alk lignin (Mn: 762 Dalton, Mw: 7660 Dalton) and the lowest in case of
WS SE Acid (Mn:588 Dalton, Mw: 3018 Dalton) and WS SE Alk lignin (Mn:569 Dalton, Mw:
3602Dalton). It is interesting, that most of the lignin fractions extracted from steam
exploded material with acid and alkaline treatment showed higher molecular weights
comparing to lignin fractions obtained from non-pretreated feedstocks (Table 3.7).
Similar findings were found by Li et al., (2009) and Maniet et al., (2017). There are two
hypotheses that could explain these results. The first is, that a small portion of low lignin Commented [AS[62]: It is also interesting that corn samples
showed greatest increase in polydispersity with steam explosion
molecular weight could be extracted during steam explosion treatment in the liquid pretreatments- this suggests that corn lignin favours condensation
reactions under these conditiions
phase (Acid Soluble Lignin) resulting in a global increase in lignin molecular weight in
solid pre-treated biomass. In turn, steam explosion process induces simultaneous effects
on physico-chemicals properties of lignin and molecular weight increases depending on
steam explosion severity factor (a measure of pre-treatment harshness to the biomass).
It has been observed, that depolymerization influenced by organics acids released during
a vapocracking step, induces the formation of carbonium -ions on Cα, which break β-O-
4 bonds and results in a depolymerization of lignin. Secondly, these ions can react with
C2 and C6 of phenolic ring and generate some polymerization. These opposite reactions
happen simultaneously and depend on physico-chemical and thermal environments that
could explain the molecular weight increase observed in grass lignin at low severity factor Commented [AW[63]: Not sure what you mean- mild steam
explosion conditions?
(Maniet et al., 2017). It’s worth mentioning, that condensation reactions are typical for
alkaline treatments and this is most likely why CS, MG and WS lignin fractions extracted
from SE Alk exhibited higher average molecular weight and polidyspersity comparing to
acid treated lignin. The highest average molecular weight and polidyspersity observed in
case of CS SE Alk indicated, that CS lignin fraction is very susceptible to condensation
reactions under these conditions. The molecular weight distribution curves in WS lignin
fractions, similarly to CIMV lignin (also WS lignin) were unimodal. In turn, the molecular
weight distribution curves of MG SE Acid lignin, MG SE Alk lignin, MG lignin CS SE Acid
lignin, CA SE Alk lignin and CS lignin were bimodal (Figure 3.16). This shows, that for these
fractions there were two distinct pools of lignin with different amounts and different
molecular weights (El Mansouri et al., 2011). It has been suggested, that small sharp
peaks that elute late indicate the presence of low molecular weight oligomers or
147
monomers (Seidel, 2008). The bimodal curves could be found in non-pretreated MG and
CS lignin, probably as the effect of partial depolymerisation during organosolv extraction.
Based on the characterisation data, it can be concluded that lignin extracted from MG,
MG SE Acid, CS SE Acid, CS SE Alk and WS SE Alk, showed very high purity (Klason lignin>
86 %, carbohydrates <3.5 % and ash content <1.5 %) (Table 3.1). Usually, lignin fractions
with a high purity, i.e low in ash and residual carbohydrates are preferred for the further
use in polymer applications. It has been shown, that a higher content of OH groups Commented [AW[64]: Don’t forget – low molecular weight and
low polydispersity also desirable-
increases the potential of lignin for use in polymer applications (for example Uniformity best so singale modal molecular weight range also best
polyurethane resins) (Chung and Washburn, 2012). Aliphatic OH groups react CORN ALK not good- HIGH POLYD/ HIGH COOH ALSO INDICATES
HIGH LEVEL OF OXIDATION REACTIONS WITH THIS FRACTION- ALSO
preferentially with the diisocyanate (Monteil-Rivera et al., 2013). High solubility of lignin FOR CS ACID LARGE INCREASE IN COOH COMPARED WITH NON-
PRETREATED- COULD SPECULATE DATA SUGGESTS THAT CORN
fractions in solvents such as dioxane or DMSO increases their application in polyurethane LIGNIN VERY SUSCEPTIBLE TO CONDENSATION REACTIONS
systems (Arshanitsa et al., 2014). According to 31P-NMR data, lignin fractions extracted
Add this for presence of shoulder
from non-pretreated MG and WS showed the highest amount of aliphatic hydroxyl
groups (>2 mmol g) (Table 3.3). MG SE Alk and WS SE Alk lignin showed the highest
solubility, in dioxane (>92 %) and DMSO (>93 %) respectively (Table 3.2). These lignin
fractions exhibit the highest potential for incorporation into polyurethanes. It is worth
mentioning, that the presence of polar groups in lignin fractions also suggests their
compatibility with polyesters (Sahoo et al., 2011). FTIR analysis showed a high intensity
of polar groups in all lignin fractions, regardless of the treatment and feedstock (Figure Commented [AW[65]: However it should be noted that this
does not diffrentiate between different classes
3.5). For the production of PF-resins, a high content of non-sterically hindered hydroxyl
groups is beneficial in combination with free aromatic rings sides on the ortho/para
positions (Gosselink et al., 2010). The reactivity of technical lignin is influenced by the
number of phenolic hydroxyl groups. This is because of the activation of the aromatic
ring in the o-position and the fact that units with free phenolic hydroxyl groups are able
to form quinone-methide intermediates, which are susceptible to nucleophilic reaction
at the benzylic carbon atom. The presence of these groups tends to increase the
reactivity of lignin towards formaldehyde when lignin is used for phenolic resins
formulation. Moreover lignin fractions with low molecular weight are preferred
candidates for PF resins as they are more reactive than high molecular weight molecules
(El Mansouri et al., 2011). According to SEC, lignin extracted from WS SE Acid material,
showed the lowest average weight molecular mass (Mn) (588) and polydispersity (5.1).
148
Lignin fractions extracted from non-pretreated CS and WS also showed relatively low
average weight molecular mass (Mn) (<710) and polydispersity (<5.4) (Table 3.7) and
could be taken into account for further incorporation to PF resins. There is no one
‘universal’ lignin, suitable for different applications. However, WS SE Acid lignin showed
a broad range of good properties. In comparison with the other lignin fractions, WS SE
Acid lignin showed the highest Klason lignin (90.06 %) content (Table 3.1), the lowest
polydispersity (5.1) and the lowest average weight molecular mass (Mn) (588) (Table
3.7). Additionally, it showed low carbohydrates impurities (2.54 %) (Table 3.1), high
solubility in dioxane (94.50 %) and DMSO (87.85 %) (Table 3.2), relatively high amount of
aliphatic OH groups (1.64 mmol g-1) and total hydroxyl groups (4.77 mmol g-1) (Table 3.3).
In view of these properties, WS SE Acid lignin could be considered a suitable raw material
for incorporation into polyesters, as well as polyurethanes or phenolic resins.
3.5 EXPERIMENT II: LIGNIN CHARACTERISATION FROM WOOD

3.5.1 RESULTS
3.5.1.1 Lignin composition
Lignin composition was analysed using protocols described in section 3.3.2.1. Table 3.8
shows the ash, sugar residues, Klason lignin (Acid Insoluble Lignin) and Acid Soluble Lignin
content (on a dry matter basis) of lignin fractions extracted from various feedstocks. The
results of the analyses showed, that source of lignin and biomass pre-treatment
influenced the content of Klason lignin and contaminants (ash and sugar). The purity of
all tested samples indicated by Klason lignin content ranged between 86.50 % - 94.17 %. Commented [AW[66]: To one decimal place
Most of the lignin fractions showed <3 % content of carbohydrates. However, the ash
content in the majority of samples exhibited >6 %. The purity of wood lignin fractions
extracted from WW, was comparable with Alcell mixed hardwoods lignin, which
exhibited a Klason lignin content of 90.61 %. In turn, all lignin fractions showed slightly
higher purity comparing to CIMV lignin standard, which exhibited a Klason lignin content Commented [AW[67]: Need to look for trends rather than
mention content of each individual sampe
of 84.56 %. The highest Klason lignin content observed was 94.17 % in the case of WW
SE Acid lignin. WW SE Acid lignin fraction also exhibited lower amount of sugar impurities
Commented [AS[68]: I left this bit for both wood and grass
analyses as I think it just mentions the lowest and the highest purity,
comparing to WW lignin, extracted from non-pretreated material. However, the amount not each individual sample, this is of high importance as based on
this mostly I pick up WS SE Acid as the lignin with the best properties
of ash reaching 6.53 % in this sample was relatively high. Lignin showing the lowest purity for polyurethanes, pf resins etc, which is one of the key findings. I
think its worth to mention it, otherwise my conclusions wont make
containing 86.50 % of Klason lignin, was observed in the case of PN SE Alk lignin. too much sense.
149
However, the amount of carbohydrates impurities in this lignin was quite low, reaching
only 1.37 %. In addition, this lignin represented very low ash content (0.44 %). The
amount of sugar residues observed for non-pretreated WW and PN lignin was 3.86 %
and 2.40 %, respectively. Steam explosion under acetic and alkaline conditions decreased
the amount of sugar residues in lignin fractions extracted from WW (2.13 % for SE Acid
and 1.46 % for WW SE Alk compared with 3.86 % for WW) and PN (1.39 % for SE Acid
and 1.37 % for SE Alk compared with 2.40 % for PN) and the marked differences were
mostly seen for xylan (in WW lignin fractions), mannan (in PN lignin fractions) and glucan
(in PN and WW lignin fractions). The amount of uronic acid also dropped following steam
explosion pre-treatment. The ash content of PN lignin fractions was lower in general
compared with WW lignin fractions. WW lignin extracted from pre-treated material,
showed a decreased amount of ash content compared with non-pretreated material. In
contrast, the amount of ash in acid and alkali pre-treated PN samples increased
compared to PN lignin extracted from non-pretreated material. The highest and lowest
ash content exhibiting 11.01 % and 0.38 % was respectively observed in case of WW
lignin and PN lignin.
150
Table 4.198: Analysis of lignin extracts from non-pretreated and acid and alkali steam exploded pine and willow. Values are presented as a percentage of dry matter (DM).
Standard deviation (SD) of the mean (n=2) is 0. ND= not detected.
Composition PN PN SE Acid PN SE Alk WW WW SE Acid WWSE Alk CIMV WS Alcell mixed

lignin lignin lignin lignin lignin lignin lignin hardwoods lignin
Total Carbohydrate 2.40 1.39 1.37 3.86 2.13 1.46 3.02 0.34
composition
Arabinan 0.08 0.05 0.08 0.05 0.04 0.06 0.23 0.00
Xylan 0.26 0.20 0.15 0.94 0.60 0.06 0.67 0.10
Mannan 0.78 0.56 0.44 0.05 0.07 0.05 0.13 0.01
Galactan 0.18 0.11 0.23 0.14 0.10 0.16 0.15 0.01
Glucan 0.46 0.30 0.39 0.56 0.43 0.54 0.99 0.06
Rhamnan 0.01 0.01 0.01 0.04 0.03 0.04 0.03 0.00
Uronic acid 0.62 0.16 0.08 2.08 0.85 0.55 0.82 0.15
Total lignin 87.04 91.01 86.78 90.41 95.10 89.22 85.89 92.09
Klason lignin 86.83 90.82 86.50 89.42 94.17 88.14 84.56 90.61
Acid Soluble Lignin 0.21 0.19 0.28 0.98 0.93 1.08 1.33 1.48
Ash 0.38 6.11 0.44 11.01 6.53 6.35 N/D N/D
151
3.5.1.2 Chemical properties and structure
3.5.1.2.1 Solubility
Lignin solubility was tested as described in section 3.3.2.2. Table 3.9 shows the solubility
of lignin fractions in different organic solvents on dry matter basis. The results of the
analysis showed, that all lignin samples were generally soluble to some degree in tested
solvents. Very high solubility of lignin fractions with values >72 % were with dioxane and
DMSO. PN lignin fractions were more soluble than WW lingnin fractions with PN SE Acid
lignin exhibiting the highest solubility in dioxane (90.22 %). Acid pre-treated PN and WW Commented [AW[69]: Round up to one decimal place
lignin fractions showed >94 % solubility in DMSO. Ethyl acetate, proved to be a poor
solvent for solubilizing all tested wood lignin fractions with values ranging from 28.25 %-
54.30 %. Similar results were observed for ethanol, which exhibited lignin solubility
ranging only between 34.70 % - 46.12 %. With regard to pre-treatments, WW lignin
fractions extracted from acid and alkali treated material showed an obvious decrease in
solubility in methanol, ethanol and ethyl acetate. In turn, solubility of the same lignin
fractions was much higher in dioxane and DMSO. PN SE Acid lignin revealed higher
solubility in most of the solvents comparing to PN lignin extracted from non-pretreated
material. Alkali pre-treatment did not show an obvioius effect on PN lignin solubility in
tested solvents.
152
Table 4.209: Solubility of lignin extracts in different organic solvents. Values are presented as a percentage of dry matter (DM) with standard deviation (SD ±) of the mean
(n=3). Commented [AW[70]: Give values to one decimal place
Lignin type Methanol Ethanol Ethyl acetate Dioxane DMSO
PN 38.03 ± 0.822 46.12 ± 0.878 28.25 ± 0.951 86.44 ± 0.695 74.24 ± 0.924
PN SE Acid 53.55 ± 0.785 34.71 ± 1.514 54.30 ± 1.025 90.22 ± 0.934 94.32 ± 0.715
PN SE Alk 40.71 ± 0.775 42.35 ± 0.621 44.52 ± 0.557 86.71 ± 0.529 87.33 ± 0.932
WW 60.48 ± 0.796 41.53 ± 0.854 40.65 ± 0.723 72.28 ± 0.752 78.20 ± 0.802
WW SE Acid 45.72 ± 0.917 34.70 ± 1.026 38.37 ± 0.533 77.54 ± 1.122 94.14 ± 0.861
WW SE Alk 47.63 ± 0.531 38.20 ± 0.781 33.17 ± 0.866 78.17 ± 0.892 75.41 ± 0.886
153
3.5.1.2.2 31 P-NMR
3.3.2.3. An example of a 31P-NMR spectrum of lignin extracted from CS SE Acid is shown
in Figure 3.20. Table 3.10 shows the amount of hydroxyl groups, consisting of aliphatic
OH, condensed phenolic OH, syringyl OH, guaiacyl OH, p-hydroxyl OH, and COOH groups.
The concentration of each hydroxyl functional group (mmol g-1) was calculated on the
basis of the hydroxyl content of the internal standard and its peak area. The results of
the analysis showed, that all fractions had similar levels of total OH groups with the
highest content observed for WW SE Alk lignin which exhibited a value of 4.47 mmol g-1
and the lowest amount of 4.04 mmol g-1 observed for PN SE Acid lignin (Figure 3.21).
Aliphatic OH groups formed the largest component of total OH content with the highest
content observed for PN lignin, which exhibited a value of 2.09 mmol g-1 and the lowest
amount of 1.27 mmol g-1 observed for WW SE Acid lignin. It has been observed, that the
amount of aliphatic OH groups was much lower in the case of lignin fractions extracted
from steam exploded material. The only exception was seen in case of WW SE Alk lignin,
exhibiting higher amount of aliphatic groups comparing to lignin fractions generated
from non-pretreated and acid treated material. In turn, it is notable that levels of other
OH groups were slightly higher in some of the lignin fractions extracted from pre-treated
material (Figure 3.22). Results for cond.phen OH groups showed, that WW lignin
fractions had a higher content compared to PN fractions (0.69-0.82 vs 0.48-0.62 mmol
g-1 ). A similar observation was made for syringyl OH groups and hydroxyl OH groups with
syringyl OH and hydroxyl OH values for WW lignin ranging from 0.73-0.89 and 0.29-0.36
mmol g-1 respectively, while respective values for PN lignin fractions ranged from 0.16-
0.18 and 0.09-0.10 mmol g-1. In contrast, PN lignin fractions showed the highest content
of guaiacyl OH groups (1.02-1.22 mmol g-1) compared with WW lignin fractions (0.59-
0.66 mmol g-1). PN SE Alk lignin and PN lignin fractions, exhibited the highest and the
lowest content of COOH groups with values of 0.53 and 0.19 mmol g-1, respectively.
There was an apparent increase in COOH groups following pre-treatment particularly in
the presence of alkali. Lignin standards showed variable composition compared with
extracted lignin fractions. Kraft Indulin AT (Softwood) lignin showed a higher OH content
154
with a total value of 6.3 mmol g-1. In general aliphatic OH, cond,phen OH, guaiacyl OH
groups were higher in this standard. P1000 (Protobind) lignin showed the highest
content of COOH groups compared to all other samples (1.05 mmol g-1) while Alcell lignin
showed the highest amount of syringylOH groups (1.05 mmol g-1) (Table 3.10).
155
Table 4.2110: Contents of functional groups as determined by 31P- NMR. The range of OH groups include: aliphatic OH, cond.Phen.OH, syringyl OH, guaiacyll OH, p-Hydroxyl
OH and COOH in (mmol g-1). Standard deviation (SD) of the mean (n=2) is 0. ND= not detected.
Aliphatic Cond. Phen

Lignin Syringyl OH Guaiacyl OH p-Hydroxyl OH COOH Total
OH OH
PN 2.09 0.48 0.17 1.04 0.09 0.19 4.06

PN SE Acid 1.67 0.62 0.18 1.22 0.10 0.25 4.04
PN SE Alk 1.73 0.54 0.16 1.02 0.10 0.53 4.08
WW 1.49 0.74 0.73 0.61 0.36 0.25 4.18
WW SE Acid 1.27 0.82 0.89 0.66 0.33 0.34 4.31
WW SE Alk 1.70 0.69 0.82 0.59 0.29 0.38 4.47
P1000 (Protobind) 1.73 0.70 0.56 0.70 0.45 1.05 5.19
Alcell 1.08 0.76 1.05 0.70 0.20 0.30 4.09
Kraft Indulin AT (Softwood) 2.35 1.36 N/D 1.88 0.22 0.49 6.30
156
31
Figure 4.4020: P-NMR spectra of PN SE Acid lignin.
157
Total content of functional groups (mmol g-1)
Total functional OH groups (mmol g-1)
6
5
4
3
2
1
0
PN PN SE Acid PN SE Alk WW WW SE Acid WW SE Alk
Lignin
Figure 4.4121: Total contents of functional groups as determined by 31 P-NMR in mmol g-1. Standard deviation (SD) of the mean (n=2) is 0.
158
Functional group content
2.5
2
Aliphatic OH
1.5 Cond. Phen OH
Syringyl OH
1
Guaiacyl OH
0.5
p-Hydroxyl OH
0 COOH
PN PN SE Acid PN SE Alk WW WW SE Acid WW SE Alk
Lignin
Figure 4.4222: Contents of functional groups in different lignin fractions. Data represent aliphatic OH, cond.Phen.OH, syringyl OH, guaiacyl OH, p-Hydroxyl OH and COOH
groups in mmol g-1. Standard deviation (SD) of the mean (n=2) is 0.
159
3.5.1.2.3 FTIR
FTIR analysis was carried out following the method described in section 3.3.2.4. Table 3.4 Commented [AW[71]: Refer back to previous table- don’t
present same table twice
gives the assignment of FTIR absorption bands of lignin, based on the literature. As seen
in Figure 3.23 all spectral profiles were very similar and confirmed the ‘core’ lignin
structure. The differences were mostly seen in the intensity of bands. All lignin fractions
showed the broad band at 3460-3410 cm-1 attributed to hydroxyl groups. Bands of very
similar intensity were observed in all samples in the ranges of 2938-2842 cm-1, indicating
aliphatic groups, 1707-1691 cm-1 indicating unconjugated aldehyde/ketone groups and
1615-1600 cm-1 indicating C=C stretching of the aromatic ring. Strong vibrations in the
ranges of 1219-1211 cm-1(characteristic for C-C, C-O and C=O stretching), 1123-1110 cm-
1 (indicating aromatic C-H deformation of syringyl units; more evident in WW samples)
and 1031-1015 cm-1 (specific for C-O stretching of primary alcohols; more evident in PN
samples) were also observed for all tested samples. In addition, strong vibrations in the
range of 1460-1459 cm-1, indicating C-H bending of methyl and methylene groups, were
evident in the spectra of all lignin fractions. Spectra showing aromatic skeleton
vibrations in the range of 1515-1511 cm-1 were observed in all samples but with
increased intensity in PN lignin fractions. C-H deformation in lignin in the range of 1425-
1423 cm-1 was observed in tested lignin fractions with minimal differences in intensity
between samples. The spectra of all extracted lignin samples exhibited vibrations
characteristic for guaiacyl units (1267 cm-1) and syringyl units (1326 cm-1). Peaks
indicating guaicayl unit vibrations were larger in spectra for PN lignin fractions compared
with WW lignin fractions. In contrast, willow lignin fractions showed peaks of higher
intensity for syringyl units. Peaks indicating carbonyl absorption were observed at 1660
cm-1, and only appeared as small peaks in PN lignin fractions extracted from non-pre-
treated and acid pre-treated material showing, that these fractions contained higher
amounts of conjugated carbonyl groups, comparing to lignin extracted from PN SE Alk
and both: non-pretreated and pre-treated WW material. Differences were also observed
at 1167 cm-1, indicating that the content of ester groups was higher in PN lignin fractions
compared with WW lignin fractions, regardless of treatment. The band at 854 cm-1,
indicating C-H out of plane of guaiacyl units was only observed in PN lignin fractions.
160
Peaks were observed at 1367 cm-1 (originating from aliphatic C-H in methyl groups) for
all lignin fractions but these were more intense in PN samples. Common to all the lignin
fractions were weak vibrations at wave lengths 835-815 cm-1 (indicating C-H bending of
syringyl+guaiacyl units) and 911 cm-1 (originating from C-H bending of syringyl units).
No obvious peaks were observed at 983 cm-1 which originate from CH=CH groups (Figure
3.23). A comparison of lignin fractions extracted from pre-treated and non-pretreated
materials showed, that the peaks in the fingerprint regions were very similar (Figure
3.24). The differences were mostly observed in the intensity of peaks. A detailed analysis
of the effect of pre-treatment on the chemical structure of lignin is described below.
WW lignin
WW lignin spectra showed highest intensity peaks in the range of 1123-1110 cm-1
(related to aromatic C-H egradation of syringyl unit). All lignin fractions showed very
similar peak intensities in the ranges 1031-1015 cm-1 (related to the C-O stretching of
primary alcohols), and 1425-1423 cm-1 (related to C-H deformation in lignin). WW SE
Acid lignin and WW lignin showed very similar profiles, for example: in the ranges 1515-
1511 cm-1 (indicating C=C stretching of aromatic ring in lignin), 1460-1459 cm-1
(indicating C-H bending of methyl and methylene groups), 1326 cm-1 (indicating C=O
stretching of the syringyl unit), 1219-1211 cm-1 (indicating C-C, C-O and C=O stretching
of guaiacyl unit), 911 cm-1 (indicating C-H bending of syringyl units, aromatic rings) and
835-811cm-1 (indicating C-H bending of syringyl plus hydroxyl units). These lignin Commented [AW[72]: Keep to same format
fractions showed more intense peaks in general compared to WW SE Alk lignin.

However, some exceptions were observed. Unconjugated carbonyl stretching of
aldehyde/ketone groups in the range of 1707-1691 cm-1 was particularly instense for
WW SE Alk lignin comparing to WW SE Acid lignin and WW lignin. Also, a higher intensity
peak at 1167 cm -1 indicating C-O stretching of ester groups was observed in case of WW
SE Acid lignin and WW SE Alk lignin comparing to lignin fraction extracted from non-
pretreated material (Figure 3.25).
161
PN lignin
FTIR spectra for lignin extracted from PN showed highest intensity peaks in the range of
1031-1015 cm-1 (related to the C-O stretching of primary alcohols). All lignin fractions
showed very similar peak intensities in the ranges of 1707-1691 cm-1 (related to
unconjugated carbonyl stretching of aldehyde/ketone groups), 1515-1511 cm-1
(indicating C=C stretching of aromatic ring in lignin), 1460-1459 cm-1 (indicating C-H
bending of methyl and methylene groups), 1219-1211 cm-1 (characteristic for C-C, C-O
and C=O stretching), 1267 cm-1 (indicating C=O stretching of guaiacyl unit) and 1123-
1110 cm-1 (related to aromatic C-H degradation of syringyl unit). PN lignin and PN SE
Acid lignin showed very similar profile for the peak occuring at 1167 cm-1. PN lignin
showed markedly higher intensity of peaks in the ranges 1031-1015 cm-1 (related to the
C-O stretching of primary alcohols) comparing to other lignin fractions. In turn, PN lignin
revealed lower intensity of peaks in the ranges 835-815 cm-1 (indicating C-H bending of
syringyl + hydroxyl units) and 854 cm-1 (indicating C-H out of plane of guaiacyl units).
(Figure 3.26).
Comparison of lignin standards (CIMV and Kraft), with extracted lignin fractions (PN SE
Acid lignin as an example) showed similarity in structure with CIMV lignin. The
differences between PN SE Acid lignin and CIMV were mostly observed in the intensity
of the peaks. Peaks in the ranges 1707-1691 cm-1 (indicating unconjugated carbonyl
stretching of aldehyde/ketone groups), 1267 cm -1 (indicating C=O stretching of guaiacyl
unit), 1167 cm -1 (indicating C-O stretching of ester groups) and 1085 cm-1 (indicating C- Commented [AW[73]: I don’t think this is the case- also look at
835- labelled differently on both spectra and again looks high in PN
O deformation in secondary alcohols and aliphatic ethers) showed much higher intensity lignin
in PN SE Acid lignin. In turn, the bands in the ranges 835-815 cm -1 indicating C-H bending
of syringyl + hydroxyl units showed slightly higher intensity in case of CIMV lignin.
Comparing to PN SE Acid lignin fraction, a band at 983 cm -1 (which originates from
CH=CH groups) was only observed in the case of CIMV lignin. In contrast, Kraft lignin
showed large differences with the structure of PN SE Acid lignin. The characteristic bands
in the range of 1707-1691 cm-1 indicating unconjugated carbonyl stretching of
aldehyde/ketone groups which gave rise to a prominent peak with the PN sample was
largely absent in the Kraft spectrum. Also, peaks within the typical band in the range of
1515-1511 cm-1 indicating C=C stretching in the aromatic ring and at 1267 cm-1 indicating
162
C=O stretching of the guaiacyl unit were largely reduced in the Kraft spectrum. A
characteristic band at 1085 cm -1, (indicating C-O deformation in secondary alcohols and
aliphatic ethers) and at 854 cm -1 (indicating C-H out of plane in guaiacyl unit)s was
observed with both Kraft lignin and PN SE Acid lignin (Figure 3.27).
S/G ratio
The S/G ratios were calculated using normalized FTIR spectra with intensity of peaks at
1327 cm-1 and 1268 cm-1 according to the method of Sammons et al. (2013) . Table 3.11
shows the results for S/G ratio in different lignin fractions. In the case of lignin fractions
extracted from PN, S/G ratios were very similar ranging from 0.39-0.42. Ratios for WW Commented [AW[74]: Ranking not important- values
lignin fractions were also very close in value (0.78-0.79) and almost double values for PN
samples (Table 3.11).
Table 4.2211: S/G ratio based on FTIR method.
Lignin sample S/G ratiio (FTIR)
WW SE Acid 0.79
WW 0.78
WW SE Alk 0.79
PN SE Acid 0.42
PN 0.39
PN SE Alk 0.41
163
Figure 4.4323: FTIR spectra of all extracted lignin fractions.
164
A
Figure 4.4424: FTIR spectra of lignin fractions extracted from treated (SE Acid, SE Alk) and non-pretreated material: A; WW, B; PN.
165
Figure 4.4525: FTIR spectra of WW lignin fractions extracted from steam exploded (SE Acid, SE Alk) and non-pretreated materials.
166
Figure 4.4626: FTIR spectra of PN lignin fractions extracted from steam exploded (SE Acid, SE Alk) and non-pretreated materials.
167
Figure 4.4727: FTIR spectra of PN SE Acid lignin (example) and lignin standards (CIMV, Kraft lignin).
168
3.5.1.2.4 TGA
Thermal stability, decomposition, temperature and ash content of all lignin samples were
determined according to the protocol presented in section 3.3.2.5. TG curves revealed
the weight loss of substances in relation to the temperature of thermal degradation,
while the first derivative of that curve (DTG) showed the corresponding rate of weight
loss. All lignin samples revealed 62.98 % - 67.10 % DM volatile content and 18.83 % -
33.24 % DM fixed carbon content. The ash content ranged between 0.38 % - 11.01 %
(Table 3.12) As can be seen from the TGA/DTG curves, all lignin fractions showed similar
trends (Figures 3.28 and 3.29). The initial weight loss, was due to water evaporation
(Wang et al., 2014). From 130 oC to 450 oC, the degradation was due to the breakage of
C-C linkages between the monomeric phenols causing structural rearrangements and
evaporation of phenolic compounds (Wörmeyer et al., 2011). The peak temperature
(Tmax) corresponding to the maximum decomposition rate, occurred in the range of
390 oC -414 oC, with the lowest temperature being observed for WW lignin and the
highest for PN SE Acid lignin (Table 3.12). Pyrolytic degradation in this region involves
fragmentation of inter-unit linkages releasing monomeric phenols into the vapour phase
and above 500 oC the process is related to the decomposition of some aromatic rings
(Tejado et al., 2007). The weight loss of lignin fractions slowed at temperatures >500 oC
and at temperatures >700 oC, the weight loss of the lignin fractions reached plateau
(Figures 3.28 and 3.29). The main differences between all degradation curves appeared
in the range of 200 oC -300 oC (Figure 3.29). A shoulder, was observed with WW SE Alk,
PN SE Alk , PN SE Acid and WW SE Acid lignin fractions (Figures 3.31 and 3.32). The
weight loss profile depended both on lignin species source and pre-treatment. As it can
be seen in Figure 3.33 which shows second derivative (DTG) curves, difference between
lignin fraction from pre-treated and non-pretreated materials in the range of 200 oC -
300 oC were observed but not substantial.
169
Table 4.2312: Proximate analysis of lignin samples determined from TGA graphs. Data represent max
temperature (Tmax) as well as volatiles, fixed carbon and ash as percentage of dry matter (DM).
Lignin type Volatiles Fixed Carbon Ash Tmax
WW SE Acid 62.98 27.69 6.53 404.36

WW 67.10 18.83 11.01 390.32
WW SE Alk 65.31 26.46 6.35 409.06
PN SE Acid 65.46 26.93 6.11 413.60
PN 66.33 31.25 0.38 405.76
PN SE Alk 64.56 33.24 0.44 412.16
170
120 120
100 100
80 80
Mass loss (%)
WW SE Acid lignin
60 60 PN SE Acid lignin
PN lignin
WW lignin
40 40 PN SE Alk lignin
WW SE Alk lignin
20 20
0 0
0 100 200 300 400 500 600 700 800 900 1000
Temp 0 C
Figure 4.4828: Thermogravimetric (TG) curves of WW and PN lignin fractions extracted from steam exploded (SE Acid, SE Alk) and non-pretreated material.
171
120
100
Derivative weight (%/0 C)
80
WW SE Acid lignin
60 PN SE Acid lignin
PN lignin
WW lignin
40 PN SE Alk lignin
WW SE Alk lignin
20
0
90 190 290 390 490 590 690 790 890
Temp oC
Figure 4.4929: Differential thermogravimetric (DTG) curves of WW and PN lignin fractions extracted from steam exploded (SE Acid, SE Alk) and non-pretreated material.
172
100 100
90 WW lignin 90
80 80
70 70
Mass loss (%)
PN lignin
60 60
50 50
40 40
30 30
20 20
10 10
0 0
0 100 200 300 400 500 600 700 800 900 1000
Temp °C
Figure 4.5030: Thermogravimetric (TG) and Differential thermogravimetric (DTG) curves of WW and PN lignin fractions extracted from non-pretreated material.
173
100 100
90 90
WW SE Acid lignin
80 80
70 70
Mass loss (%)
60 60
PN SE Acid lignin
50 50
40 40
30 30
20 20
10 10
0 0
0 100 200 300 400 500 600 700 800 900 1000
Temp °C
Figure 4.5131: Thermogravimetric (TG) and Differential thermogravimetric (DTG) curves of WW and PN lignin fractions extracted from steam exploded material with acid
treatment (SE Acid).
174
Figure 4.5232: Thermogravimetric (TG) and Differential thermogravimetric (DTG) curves of WW and PN lignin fractions extracted from SE material with alkaline treatment (SE
Alk).
175
90
AA
70
Sec.Der.g2/°C2
50 WW lignin
30
10 PN lignin
-10 100 150 200 250 300 350
-30
-50
Temp °C
90
B
B
70 WW SE Acid lignin
50
Sec.Der.g2/°C2
30
10 PN SE Acid lignin
-10 100 150 200 250 300 350

-30
-50
Temp °C
C 90
C
70
Sec.Der.g2/°C2
50
30 WW SE Alk lignin
10
-10 100 150 200 250 300 350 PN SE Alk lignin
-30
-50
Temp °C
Figure 4.5333: Second derivative DTG curves of lignin fractions extracted from different materials (pre-
treated with steam explosion and non-pretreated): A; non-pretreated, B; SE Acid, C; SE Alk.
176
3.5.1.2.5 Molecular weight distribution (SEC)
The molecular weight distribution of lignin samples were obtained by Size Exclusion
average (Mn) and weight average (Mw) molecular weights and polydispersity (Mw/Mn)
of the all lignin samples are shown in Table 3.13. As can be seen below, the Mn, Mw and
Mw/Mn ranged from 487-946 Dalton, 2144-9875 Dalton and 4.4-10.4, respectively. All
lignin fractions extracted from steam exploded material acid and alkaline treated
showed a lower molecular weight compared with non-pretreated samples. The highest
values of average weight molecular masses were found for WW lignin (Mn: 946 Dalton,
Mw: 9875 Dalton) and the lowest for PN SE Acid lignin (Mn:487 Dalton, Mw: 2144
Dalton). WW lignin fractions showed higher average molecular masses (Mn:756-946) Commented [AW[75]: Better not to rank
Commented [AS[76R75]: I left it as well for grasses, as based
compared with PN fractions (Mn:487-543). Most lignin fractions showed a comparable on it this one sentence i pick up lignin suitable for PF resins
degree of polydispersity (Mw/Mn: 4.4-6.3), which was comparable with Alcell lignin
(Mw/Mn: 4.3), with the exception of WW lignin (Mw/Mn: 10.4). Also, excluding WW
lignin, all lignin samples showed lower polydispersity than CIMV lignin (Table 3.13). The
molecular weight distribution curves for PN lignin fractions were unimodal.In contrast,
the molecular weight distribution curves for WW lignin fractions were bimodal.
Differences between peaks for lignin fractions extracted from SE Acid, SE Alk and non-
pretreated materials were observed (Figure 3.34). The molecular weight distribution of
PN lignin fractions was very similar to CIMV lignin showing unimodal curve (Figure 3.35).
In contrast, the molecular distribution of WW lignin fractions was bimodal, showing the
presence of low-molecular weight fractions in common with Alcell lignin (Figure 3.36).
177
Table 4.2413: Average molar mass data (Mw, Mn) and polydispersity (Mw/Mn) of all lignin samples.
WW SE Acid 898 5450 6.1

WW SE Alk 756 3616 4.8
WW 946 9875 10.4
PN SE Acid 487 2144 4.4
PN SE Alk 504 2386 4.7
PN 543 3448 6.3
CIMV lignin 907 8602 9.5
Alcell 681 2948 4.3
Lignin type SEC elution profiles
WW SE Acid
WW SE Alk
WW
178
PN SE Acid
PN SE Alk
PN
CIMV
Alcell
Figure 4.5434: Size Exclusion Chromatography (SEC) profiles of wood lignin fractions.
179
0.8
0.7
0.6
0.5 PN SE Alk lignin

PN lignin
0.4
PN SE Acid lignin
AU
0.3 CIMV
Alcell
0.2
0.1
0
15 16 17 18 19 20 21 22 23 24 25
-0.1
Minutes
Figure 4.5535: SEC profiles of PN SE Alk, PN SE Acid and PN lignin fractions comparing to lignin standards. Commented [AW[77]: Wrong species
180
0.8
0.7
0.6
WW SE Acid lignin
0.5
WW lignin
0.4
AU
WW SE Alk lignin
0.3
CIMV
0.2
Alcell
0.1
0
15 16 17 18 19 20 21 22 23 24 25
-0.1
Minutes
Figure 4.5636: SEC profiles of WW SE Alk, WW SE Acid and WW lignin fractions comparing to lignin standards. Commented [AW[78]: Wrong species
181
3.5.2 DISCUSSION
Extensive efforts have already been made to explore lignin application in the field of
biomaterials (Belgacem and Gandini, 2008; Lora, 2008; Stewart, 2008; Doherty et al.,
2011). They can be used as green components in thermoplastics, or as a reagents in
polyurethanes, polyesters, epoxy or phenolic resins (Monteil-Rivera et al., 2013).
However, the physico-chemical properties of lignin, and therefore its suitability for
specific applications, are largely dependent on the plant species and the isolation
processes (Belgacem and Gandini, 2008). As seen in section 3.4, lignin fractions extracted
from grasses and residual cereal biomass, using the organosolv process and combined
steam explosion-organosolv process exhibited a range of different properties showing
potenital for incorporation in the production of polyurethanes, phenolic resins or even
polyesters. Therefore, comprehensive analysis of softwood and hardwood lignin
fractions was also merited in order to assess, which material application is most suited
for the different types of wood lignin.
General composition analysis typically distinguishes between the Klason lignin, Acid
Insoluble Lignin (AIL), Acid Soluble Lignin (ASL), residual carbohydrates and ash. In this
study, most lignin samples showed relatively high purity, containing over 70 % Klason
lignin and <5 % of carbohydrates (Xu et al., 2006). WW lignin (hardwood) revealing 89.42
% of Klason lignin, showed similar purity to Alcell mixed hardwood lignin, exhibiting
Klason lignin content of 90.61 %. In turn, the purity of all lignin samples (>86 %) was
slightly higher comparing to CIMV WS lignin, revealing Klason lignin content of 84.56 %
(Table 3.8). However, the feedstock source and conditions used for lignin extraction in
this research were different, which could have affected the final composition. The high
amount of sugar impurities in CIMV WS lignin (3.02 %) indicated a strong association of
hemicellulose and lignin in this grass species and the issue of covalent bonds between
lignin, hemicellulose and cellulose has been previously reported in the literature
(Monteil-Rivera et al., 2013). Comparing to other lignin fractions, WW lignin showed
higher carbohydrates content than CIMV lignin with relatively high levels of xylan and
glucan as well as uronic acid indicating presence of hemicellulose and pectins
respectively. While xylan was not observed in PN samples, higher contents of mannan
182
were detected what also indicates presence of hemicellulose. In turn, the amount of
sugar residues in all lignin fractions was higher comparing to Alcell mixed hardwood
lignin, exhibiting only 0.34 % (Table 3.8) . According to Huijgen et al., (2014), Commented [AW[79]: I would just say here that extracted
lignin was of comparable purity with standard lignin samples.
carbohydrate impurities could partly originate from residual organosolv liquor, adhering
Willow lignin has more carbohydrates than cimv,
Interestingly WW lignin had a high content of uronic acid which
to the precipitated and sedimented lignin. It is interesting, that steam explosion with acid suggests presence of pectin in these samples. This fraction also
contains relatively high xylan and glucan.
treatment increased Klason lignin content in both: pine and willow lignin fractions. The In contrast to other fractions PN lignin contains a relatively high
mannan component –
highest purity, exhibiting 94.17 % of Klason lignin was seen in case of WW SE Acid lignin.
In general Acid/Alk SE reduces uronic acid- alk se reduces xylan
Pre-treated lignin fractions also revealed lower amounts of carbohydrate impurities Commented [AW[80]: I would leave this out
comparing to non-pretreated lignin fractions (Table 3.8). This is most probably because
a large proportion of the sugars were removed during steam explosion (unpublished
data). Lignin extracted from non-pretreated and alkali pre-treated softwood (PN),
showed the very low ash contents (0.38 % and 0.44 % respectively) (Table 3.8). These
results were in close agreement with data presented by Pan and Sano, (2005) who
showed, that ash content in wood plants is much lower than in herbaceous plants and
exhibits <1 %. In contrast, the ash content of lignin extracted from willow was relatively
high ranging from 6.35 %-11.01 % (Table 3.8). These results did not agree with data
presented by Nitsos et al., (2016) and Hosseinaei et al., (2016), who reported organosolv
hardwood lignin fractions with the ash content <1%. A possible factor is that a different
organosolv process (formic/acetic acid) was used in this study, which could have affected
final ash content in willow lignin fractions. It has previously been observed, that loss of
organic volatiles in harsh conditions (such as acid, alkali, higher temperature, longer
residence time) is the main reason increasing ash content (Mukherjee and Borthakur,
2004; Horn et al., 2011). This may also account for the high ash value observed in the
acid pre-treated PN lignin fraction. It is interesting, that steam explosion under acid and
alkaline treatment notably decreased the amount of ash in WW lignin (Table 3.8). This Commented [AW[81]: Don’t say significantly unless you use
statistics
could possibly be due to the ash content being removed during the pre-treatment.
Similar results were presented by Han et al., (2010), who also reported decreased ash
and silica content after steam explosion.
Analysis of the solubility of lignin fractions in different organic solvents is also very
important, because it is known, that lignin fractions have limited solubility in organic
solvents, mostly due to the heterogeneous three-dimensional structure (El-Khaldi
183
Hansen et al., 2016). The result of the comprehensive analysis carried out in this study
showed, that dioxane and DMSO were the best solvents exhibiting respectively >72 %
and >74 % solubility of all tested lignin samples (Table 3.9). Based on the Hildebrand
theory, it is predicted that when the solubility parameters of lignin and the solvent are
the same, the ability to dissolve the lignin will increase. Therefore, DMSO with a similar
solubility parameter as lignin is proposed to be one of the best solvents (Sameni et al.,
2017). It is worth mentioning, that results presented in this study were in close
accordance with Cybulska et al., (2012), who demonstrated very high solubility of
organosolv lignin samples in dioxane. Higher or lower solubility of lignin fractions
obtained from steam exploded material (both with acid and alkaline treatment) could be
due to presence or absence of impurities or changes in molecular weights. Generally
speaking, lignin with higher molecular weight reveals lower solubility in organic solvents
(Sameni et al., 2017). This pattern was observed with tested PN lignin fractions (Tables
3.9 and 3.13) where a drop in molecular weight corresponded with an increase in
solubility of acid and alkali pre-treated fractions. This may also partly explain the notably
lower solubility of higher molecular weight WW lignin fraction in dioxane and DMSO. As
seen in this study, PN lignin fractions extracted from acid and alkali treated material
showed an obvious increase in solubility in most of the tested solvents. Higher solubility
in dioxane and DMSO was also observed for WW lignin extracted from SE Acid and SE Alk
material. This was consistent with lignin composition results, which revealed much lower
amount of sugar impurities in these fractions (Tables 3.8 and 3.9). Sameni et al., (2017)
also states, that lignin solubility can relate to number of aliphatic hydroxyl groups. The
results presented in this study were in close accordance with outcomes presented by
this author. Higher solubility was observed for lignin fractions generated from pre-
treated PN revealing lower amount of aliphatic groups comparing to lignin fractions
extracted from non-pretreated feedstocks (Tables 3.10 and 3.13). Although, different
pattern were likely to occur, it was suggested, that the ability of a solvent to dissolve a
variety of isolated lignin fractions can be increased by increasing the hydrogen-binding
capacity of the solvent. This is because the hydrogen bonding is a major molecular
interaction between hydroxyl groups of lignin and organic solvents (Horvarth, 2005).
184
31P-NMR spectroscopy technique has been established to be a very powerful tool for the
characterization of phenolic hydroxyl (p-hydroxyphenyl, guaiacyl, and syringyl
structures), aliphatic hydroxyl and carboxylic acid groups present in the lignin samples
(Cateto et al., 2008). Results of the analysis carried out in this study showed, that
aliphatic OH groups showed the highest content of the total OH groups observed
(highest observed value of 2.09 mmol g-1 in PN lignin) (Table 3.10/Figure 3.22). It has
been proposed, that acetic and formic acid treatments tend to liberate β-O-4 bonds and
resulting lignin fractions with high levels of OH aliphatic groups (Villaverde et al., 2010).
It is interesting, that apart of WW SE Alk lignin, the amount of aliphatic groups markedly
decreased and the amount of phen.cond. groups increased, following steam explosion
pre-treatment (Table 3.10). Similar findings have previously been reported by Maniet et
al., (2017). A reduction in the amount of aliphatic groups could be linked to do decrease
in sugar residues which was evident in pre-treated PN and WW lignin fractions (Table
3.8) (Huijgen et al., 2014). The higher content of COOH in most of the akali pre-treated
lignin fractions possible relates to oxidation, observed in this type of pre-treatment (Sun
et al., 2014). All lignin samples showed very similar results to the lignin standard P1000
and Alcell lignin. Kraft Indulin AT (Softwood) lignin showed a distinct profile with higher
aliphatic, cond.phenolic, and guaiacyl OH groups compared to other lignin fractions.
Interestingly COOH group content was markedly higher in P1000 (Protobind) lignin
compared with other samples. Syringyl and guaiacyl OH contents showed a predictable
pattern with syringyl groups showing a higher content in hard wood samples (WW lignin,
Alcell lignin) and guaiacyl groups showing higher content in PN lignin and Kraft lignin
samples (Table 3.10). In general softwood lignin such as Kraft Indulin AT reveal high
amount of guaiacylOH groups, typical for this type of feedstock. In turn, the presence of
syringyl OH groups is typical for hardwood lignin such as Alcell lignin (Jingjing, 2011).
The FT-IR spectra for all organosolv lignin fractions were very similar within species,
confirming that the "core" of lignin structure does not change dramatically during the
various treatments. (Figures 3.23 and 3.24). Lignin fractions generated in this study
showed very similar structure to CIMV lignin. The differences were mostly seen in the
intensity of the bands. In contrast, lignin samples showed marked difference when
compared to Kraft lignin. The presence of peaks in the range of 1707-1691cm-1
185
(indicating unconjugated carbonyl stretching of aldehyde/ketone groups) has been
shown to be characteristic for organosolv lignin fractions, and originate from carbonyl
groups released during acid hydrolysis which may explain why it is not observed in the
Kraft lignin sample (Tejado et al., 2007). However, the presence of bands at 1085 cm-1, Commented [AW[82]: check
(indicating C-O deformation in secondary alcohols and aliphatic ethers) and at 854 cm -1
(indicating C-H out of plane in position 2,5 and 6 of G units, typical for Kraft lignin (Li et
al., 2009) was also observed in PN lignin fractions (Figure 3.27) which is consistent with Commented [AW[83]: not just one shown in figure
Kraft lignin being extracted from the same feedstock. Lignin fractions extracted from Commented [AW[84]: full name
WW and PN revealed a mean S/G ratio of : 0.79 and 0.41 respectively (Table 3.11). These Commented [AW[85]: caluclate average pn and ww three
values
results slightly differed from data presented in the literature. According to Hosseinaei et
al., 2016, S/G ratio of organosolv hardwood lignin varied between 3.12 to 5.90
depending on the process conditions. In turn, research carried out by Nicholson et al.,
(2016) revealed, that S/G ratio of 5 different hardwood lignin fractions varied between
1.23-1.97 depending on feedstock source. However, the type of organosolv and lignin
source analysed in this study was different comparing to those presented by authors
above, what could affect the final composition of lignin. Although, there are no reports
confirming S/G ratio in softwood lignin it is clear, that the ratio will be higher in hardwood
than in softwood feedtocks.
TGA was used to determine thermal stability of lignin fractions investigated in this study.
As can be seen from the TGA/DTG curves, all lignin fractions showed very similar trends
(Figures 3.28 and 3.29). The specific shoulder seen in WW lignin extracted from SE Acid
material (Figure 3.31) and PN and WW lignin fractions extracted from SE Alk (Figure 3.32)
showed the possible existence of a thermally less stable domain within the lignin polymer
(Toledano et al., 2011). That could potentially indicate the degradation of a
polysaccharide component. Hemicellulose, is structurally amorphous, heterogeneous in
composition and may contain different combination of arabinose, galactose, mannose,
xylose and decomposes mainly between 220 oC - 315 oC (Liu et al., 2014). The Commented [AW[86]: PAPER BELOW IS WORTH A LOOK
BECAUSE IT BREAKS DOWN PARTS OF CURVE INTO DIFFERENT
COMPONENTS
thermolysis reactions of polysaccharides occur through dehydration, decarboxylation,
THERMOGRAVIMETRIC ANALYSIS AND KINETIC STUDY OF BAMBOO
decarbonylation and the cleavage of glucoside bonds, C-H, C-O and C-C bonds (Sahoo et WASTE TREATED BY ECHINODONTIUM TAXODII USING A MODIFIED
THREE-PARALLEL-REACTIONS MODEL
al., 2011). The presence of shoulder, indicating polysaccharide components was already Chen et al. Bioresource technol-
observed by Yu et al., (2015). It is worth mentioning, that the presence of shoulders
186
could also be caused by changes in the chemical composition of lignin fractions occurring
in the process of steam explosion. During steam explosion with acid treatment, some
fractions of the hemicellulose and cellulose are hydrolysed. After the condensation and
recondensation reactions, combining lignin and other degradation products might create
a so-called ‘pseudolignin’. This phenomenon has been previously observed with
feedstocks pre-treated with acids (Sannigrahi et al., 2011). However, according to recent
studies, ‘pseudolignin’ was also observed in feedstocks which were steam exploded with
alkaline treatment. Based on these observations, it’s very likely, that significant irregular
peaks at temperatures between 200 oC -300 oC could be a signal of ‘pseudolignin’.
The number average molecular mass (Mn), weight average molecular mass (Mw) and
polydispersity (PD) of the lignin macromolecule, were determined by Size Exclusion
Chromatography (SEC). The results of the analysis showed, that the number average Commented [AS[87]: I left it as well for grasses, as based on it
this one sentence i pick up lignin suitable for PF resins
molecular mass (Mn) in case of WW lignin fractions (756-946) was higher comparing to
PN lignin fractions (487-543). The highest values of average weight molecular masses
were determined in case of WW lignin (Mn: 946 Dalton, Mw: 9875 Dalton) and the
lowest in case of PN SE Acid (Mn:487 Dalton, Mw: 2144 Dalton). It is interesting, that all Commented [AW[88]: Here say average weight for each
species and that higher in case of willow -average of 3
lignin fractions extracted from steam exploded material with acid and alkaline treatment
showed lower molecular weight comparing to lignin fractions obtained from non-
pretreated feedstocks (Table 3.13). Similar findings were found by Li et al., (2009) and
Maniet et al., (2017). It has been reported, that Mw and Mn decrease when the pre-
treatment severity factor is very low or very high. Study carried out by Maniet et al.,
(2017) showed, that low molcelular weight lignin fractions were generated at severity
factor 1.4. In turn, an obvious increase in molecular weight was observed with the
severity factor 2.6-3.8. At severity factor higher than 3.8, low molecular weight lignin
fractions were generated again. According to Nitsos et al., (2016), an increase in
molecular weight for lignin extracted from non-pretreated feedstocks can also be
caused by impurities such as carbohydrates, that can increase the signal in high
molecular weight regions. After removing these carbohydrate regions by enzymatic Commented [AW[89]: Need to explain CHECK THIS
hydrolysis, a decrease Mn is observed. As seen in section 3.5.1.1, the amount of sugar

impurities in lignin markedly decreased following steam explosion with acid and alkaline
treatment. That could potentially explain lower Mn in lignin fractions extracted from SE
187
material. The molecular weight distribution curves in PN lignin samples, similarly to CIMV
lignin were unimodal. In contrast, the molecular weight distribution curves of WW lignin
fractions were bimodal (Figure 3.34). This shows, there were two lignin fractions of
different abundance and different molecular weights (El Mansouri et al., 2011). It has
been suggested, that small sharp peaks that elute late indicate the presence of low
molecular weight oligomers or monomers (Seidel, 2008). The bimodal curves found in
lignin extracted from non-pretreated WW were most likely due to the effect of partial
depolymerisation during organosolv extraction.
Based on the found characterisation data, it can be concluded, that lignin fractions
extracted from PN and PN SE Alk showed very high purity (Klason lignin >86 %,
carbohydrates <2.5 % and ash content <0.5 %) (Table 3.8). Usually, lignin preparations
with a high purity, i.e low in ash and residual carbohydrates are preferred for the further
use in polymer applications. It has been shown, that a higher content of OH groups
increases the potential of lignin for use in polymer applications (for example
polyurethane resins) (Chung and Washburn, 2012). Aliphatic OH groups react
preferentially with the diisocyanate (Monteil-Rivera et al., 2013). High solubility of lignin
fractions in solvents such as dioxane or DMSO also increase their application in
polyurethane systems (Arshanitsa et al., 2014). According to 31P-NMR data, lignin
extracted from non-pretretated PN showed the highest amount of aliphatic hydroxyl
groups (>2 mmol g-1) (Table 3.10). PN SE Acid lignin indicated the highest solubility,
respectively in dioxane (>90 %) and DMSO (>94 %) (Table 3.9). These lignin fractions
exhibit the highest potential for incorporation into polyurethanes. It is worth mentioning,
that the presence of polar groups in lignin also suggests their compatibility with
incorporation into polyesters (Sahoo et al., 2011). FTIR analysis indicated the high
concentration of polar groups in all lignin fractions, regardless the treatment and
feedstock (Figure 3.23). For the production of PF-resins, a high content of non-sterically
hindered hydroxyl groups is beneficial in combination with free aromatic rings sides on
the ortho/para positions (Gosselink et al., 2010). The reactivity of technical lignin
fractions is influenced by the number of phenolic hydroxyl groups. This is because of the
activation of the aromatic ring in the o-position and the fact that units with free phenolic
hydroxyl groups are able to form quinone-methide intermediates, which are susceptible
188
to nucleophilic reaction at the benzylic carbon atom. The presence of these groups tends
to increase the reactivity of lignin towards formaldehyde when lignin is used in phenolic
resin formulations. Also lignin fractions with low molecular weight are preferred
candidates for PF resins. They are more reactive than those with high molecular weight
molecules (El Mansouri et al., 2011). According to SEC, lignin extracted from PN SE Acid
presented quite low low average weight molecular mass (Mn) (487 Dalton) and
polydispersity (4.4). Also, lignin fractions extracted from WW SE Alk and PN SE Alk Commented [AW[90]: Mn and polydispersity no better to
present
presented relatively low average weight molecular mass (756 Dalton and 504 Dalton, Commented [AS[91]: Why if I presented vales for NMR
respectively) and polydispersity (4.8 and 4.7, respectively) and could be considered for Commented [AW[92]: As above
further incorporation to PF resins (Table 3.13). There is no one ‘universal’ lignin, suitable
for different applications. However, PN SE Acid lignin showed a broad range of good
properties. Among all of the other lignin fractions, PN SE Acid lignin indicated very high
Klason lignin (90.82 %), the lowest amount of carbohydrates impurities (1.39 %) (Table
3.8), the lowest polydispersity (4.4) and the lowest molecular weight (487 Dalton) (Table Commented [AW[93]: As above
3.13). Additionally, it showed high solubility in dioxane (90.22 %) and DMSO (94.32 %)
(Table 3.9) and relatively high amount of aliphatic OH groups (1.67 mmol g-1) (Table 3.10).
Thanks to these properties, PN SE Acid lignin could be a suitable raw material for
incorporation into polyesters, as well as polyurethanes or phenolic resins.
189
3.6 CONCLUSIONS Commented [AW[94]: This section is written in tne form of
abstract – best not to give values/
Say methods were effective for differenting lignin fractions- species

Lignin was extracted from grass: Miscanthus giganteus, corn stover, wheat straw as well and treatment effects observed- on this basis certain fractions
showed promising properties for various applications
as wood: pine, willow feedstocks using the organoslv process and combined steam
explosion-organosolv and the different fractions were characterised for further use in
high-value products. Methods used in this study including lignin purity analysis, solubility
analysis, FTIR, TGA, SEC and 31P-NMR gave a comprehensive analysis of lignin
characteristics. Lignin composition analysis revealed, that all lignin samples were
relatively pure and generally soluble to some degree in different organic solvents. FTIR
analysis highlighted the fact, that steam explosion pre-treatment did not markedly
change the core lignin structure. The TGA/DTG curves of all lignin fractions also showed
very similar trends. However, it has been reported, that lignin fractions generated from
acid or alkali treated grass feedstocks exhibited higher molecular weight. In turn, lignin
fractions extracted from wood feedstocks revealed lower molecular weight. Both grass
and wood lignin fractions produced from steam exploded material showed higher
amount of total OH groups and the presence of ‘pseudolignin’, most likely as a result of
condensation reactions occurring during these conditions. The overall assessment
revealed some differences in lignin composition and properties between species and
pre-treatment processes. On this basis certain fractions showed promising properties for
various applications.
190
Chapter 4
191
Chapter 4: DEVELOPMENT OF NEW METHODS FOR SCREENING
ORGANOSOLV LIGNIN METABOLISING ORGANISMS
4.1 ABSTRACT
Miscanthus giganteus and CIMV organosolv lignin were treated with selected fungal and
bacterial strains, to determine the ligninolytic capability of microorganisms. The main
aim of this chapter was to develop new methods for the screening of fungi and bacteria
capable of metabolising different forms of lignin in order to increase their suitability for
synthesis of high-value products. Filamentous fungi:Trichoderma harzianum and gram Commented [AW[95]: Give full latin name
negative bacteria: Pseudomonas putida, Pelomonas saccharophila and Pseudomonas

fluorescens were screened for their ability to grow in minimal media supplemented with Commented [AW[96]: Full latin name
different amounts of Miscanthus giganteus and CIMV lignin as the sole carbon and
energy source. Based on the positive results showing the ability of T. harzianum to utilize
lignin containing feedstock as a carbon and energy source, further experiments were
carried out only in the presence of CIMV lignin. Decolourization of methylene blue was
used to indicate the presence of ligninolytic enzymes produced by the potential lignin-
degrading strains. T. harzianum grew on YNB solid media supplemented with different
amounts of lignin: 0.25 %, 0.5 % and 1 % (w/v). Out of the three concentrations, media
supplemented with 0.25 % (w/v) of lignin and methylene blue exhibited the most
efficient dye oxidation. Oxidation of methylene blue was also observed with the spent
culture supernatants, with the highest efficiency in media supplemented with 0.25 % and
0.5 % (w/v) of lignin. None of the bacterial strains were able to grow on M9 solid media
supplemented with 0.25 % (w/v) of lignin. However, growth in liquid media under the
same conditions was observed for all microbial species. Overall, the results showed that
T. harzianum, P. putida, P. saccharophila and P. fluorescens have the ability to utilise
CIMV lignin as the sole carbon and energy source for growth. Different methods
investigated in this study helped to establish media composition, and optimal conditions
for the expression of ligninolytic enzymes. Based on preliminary tests, it has been shown,
that growth in liquid media supplemented with 0.25 % of CIMV lignin could be the basis
for screening microorganisms for application in industrial biotechnology.
Keywords: • Screening • CIMV lignin • Fungi • Bacteria• Ligninolytic enzymes
192
4.2 INTRODUCTION
Lignin can be used in lots of applications (Laurichesse and Avérous, 2013). It can be
converted to fine chemicals, building blocks for plastics and for any other bio-based
materials (Bugg and Rahmanpour, 2015). However, it has been demonstrated, that use
in its extracted form is not very effective (Laurichesse and Avérous, 2013). In many cases,
the higher end uses of lignin have not been achieved previously because of structural
complexity, thermal instability or even augmented reactivity. In order to improve this
limitation, different types of modifications of lignin have been proposed to increase its
chemical reactivity and solubility in organic solvents and to reduce the brittleness of
lignin-derived polymers (Argyropoulos, 2012).
Regarding technology for the modification of lignin, enzymatic methods are amongst the
most promising areas for the development of new processes. They aim to use different
microorganisms for the bioconversion of lignin polymers as a raw material for industrial
products (Buranov and Mazza, 2008; Asina et al., 2017). This can be achieved directly
through incubation of the microorganism with the lignin fraction or indirectly through
modification with applied enzyme.
Microorganisms which can metabolise lignin achieve this through a suite of ligninolytic
enzymes. Extensive efforts have been made to isolate and characterize ligninolytic
microorganisms. Some white-rot and brown-rot fungi were among the first identified to
be able to degrade lignin (Tian et al, 2016). Fungi depolymerize lignin via an enzymatic
procedure performed by ligninolytic enzymes including manganese peroxidase (MnP),
lignin peroxidase (LiP), versatile peroxidase (VP) and laccase (Janusz et al., 2013). In
contrast, lignin-degrading bacteria are less well studied, but several examples have been
found among a-Proteobacteria (e.g., Sphingomonas sp.), g-Proteobacteria (e.g.,
Pseudomonas sp.), and actinomycetes (Rhodococcus, Nocardia or Streptomyces sp.). Commented [AW[97]: Could include taylor 2012 ref here- see
details below
Bacterial enzymes which are recognized as being involved in lignin degradation include
laccases, ring cleaving dioxygenases and monooxygenases (Bandounas et al., 2011).
Dyp-type peroxidases, which play a similar role to fungal lignin peroxidases, as well as
vanillin dehydrogenases have also been reported (Ding et al., 2015; Taylor et al., 2012).
193
Enhancement of ligninolytic enzyme production and induction of enzyme activity are
important factors for making a lignin bioconversion process feasible for industrial
applications. Successful induction of ligninolytic enzymes requires a combination of
appropriate microorganisms, culture media and, in some instances, inducers (Asina et
al., 2017). Although many microbes and enzymes which degrade lignin have been
reported so far, they have not been optimized for industrial scale applications (Aarti et
al., 2015).
There are reports in the literature of application of laccases as a catalyst for increasing
crosslinking in fibreboard production to increase material strength (Stewart, 2008). A
study with model lignin compounds demonstrated, that a range of products could be
produced by oxidation with lignin peroxidase or laccases with different mediators.
Laccase reactions resulted in the formation of ketone or aldehyde groups while lignin
peroxidase catalysed breakdown with the generation of hydroxyl groups (Bohlin et al.,
2005).However, it needs to be noted, that studies demonstrating efficient bioconversion
of model lignin compounds cannot be directly applied to the modification and
deconstruction of industrial lignin which is significantly modified compared the native
lignin (Asina et al., 2017).
There are several studies which showed, that native lignin-degrading microorganisms
can also utilize technical lignin. With regard to bacterial species, R. erythropolis, P. putida,
and Sphingobacterium sp. have been observed to utilize Kraft lignin and CIMV
Organosolv lignin (Taylor et al., 2012; Taylor, 2013). The example of fungal species which
have demonstrated the ligninolytic cability towards Kraft lignin has previously been
reported in the case of P. chrysporium (Lundquist et al., 1977), A. fumigatus (Kadam, K.L.,
1986) and C. versicolor (Brzonova et al., 2017).
Among all bacterial strains, P. putida is currently getting increased attention in the
industrial biotechnology field as a source of enzymes for the synthesis of bio-based
polymers and fine chemicals (Poblete-Castro et al., 2012). It is a ubiquitous gram-
negative soil bacterium, which has been widely studied for its remarkable substrate-
utilizing capacity (Ravi et al., 2017). Although, P. putida already revealed the ability to
utilise technical lignin, characterization studies were limited to FTIR analysis (Taylor,
2013). Apart of P. putida, there are number of other microorganisms which show good
194
potential for lignin bioconversion. Other bacterial strain with ligninolytic potential
include P. saccharophila and P. fluorescens. These strains have been observed to
metabolise aromatic compounds (Stringfellow and Aitken, 1995; Bugg et al., 2000) and
P. fluorescens has been shown to produce Dyp-peroxidase (Bugg and Rahmanpour,
2015; de Gonzalo et al., 2016). In turn, filamentous fungi such as Trichoderma harzianum
appear to be the most efficient producers of enzymes capable of degrading
lignocellulosis feedstocks. (Jørgensen, 2003). Additionally, the ligninolytic capability of T.
harzianum assessed by its ability to utilize lignin (Rubeena et al., 2013) or lignin-related
compounds (Dabhi et al., 2017) has been also reported in literature.
The overall objective of this thesis was to produce lignin with properties for industrial
applications. Results described in Chapter 3 showed, that only 2 out of 5 species
investigated, yielded lignin fractions suited to the production of different high-value
products. That indicated, that further processing of the other lignin fractions is required
for utilisation in industry. Hence, it was postulated, that bioconversion would be a first
step towards enhancing their characteristics, for conversion into high-value chemicals as
well as building blocks for new bio-based materials. The main aim of the study presented
in this chapter was to develop new methods for screening microorganisms able to
metabolise lignin in order to improve their suitability for high-value products. The Commented [AW[98]: Comment above fits here-
filamentous fungi T. harzianum and gram negative bacteria: P. putida, P. saccharophila

and P. fluorescens were selected for this study because of the properties described
above. Furthermore, in-house bacterial strains were chosen on the basis of their ability
to metabolise aromatic compounds. These fungal and bacterial microorganisms were
screened for growth in minimal media with different amounts of Miscanthus giganteus
(lignocellulosic feedstock) or CIMV organosolv lignin (technical lignin) as the sole carbon
and energy source. Decolourisation of methylene blue dye was used as an indicator of
oxidative activity of ligninolytic enzymes produced by the potential lignin metabolizing
strains. Results of the preliminary tests were compared and discussed in the context of
optimisation of appropriate methods for screening and expression of ligninolytic
enzymes.
195
All experiments presented in this study were carried out using materials and methods
described below. The procedures used for culture development, maintenance,
screenings and assays were carried out under aseptic conditions, using sterile chemicals
and laboratory equipment. Unless otherwise stated, all plastic ware and commonly used
chemicals of the highest grade available were purchased from Fisher Scientific
(Lecicestershire, UK) or Sigma-Aldrich Company Ltd (Dorset, UK). Any glassware used in
the carried out procedures were of Duran or Pyrex brand. For all steps, purified water,
(Elga Purelab Classic UV, VWS UK Ltd, 18.2 mega ohm-cm resistivity at 25 °C) was used
unless otherwise stated.
4.3.1 FEEDSTOCKS
The feedstocks used in this study were Miscanthus giganteus and Compagnie Industrielle
de la Matière végétale (CIMV) organosolv lignin. Miscanthus giganteus (MG) was Commented [AW[99]: In full first time in chapter
collected from Aberystwyth University, UK and had been harvested in spring 2012. The
CIMV organosolv wheat straw lignin, used as a lignin standard was kindly supplied by
CIMV Company in Pomacle, Champagnes-Ardennes, France.
4.3.2 SOURCES OF MICROORGANISMS
The fungal and bacterial species used in this study were: Trichoderma harzianum,
Pseudomonas putida, Pelomonas saccharophila and Pseudomonas fluorescens. T. Commented [AW[100]: Need to include in discussion as you
just mention pseudomonas species
harzianum (coded TH), cultured on YPD agar media was kindly provided by Wageningen Commented [AW[101]: Details about strains metabolic activity
UR, Food & Biobased Research, Wageningen, The Netherlands. P. putida (NCIMB 8248;
coded PP), P. saccharophila (NCIMB 8570; coded PS) and P. fluorescens (NCIMB
9397coded PF) were supplied in glycerol stocks by IBERS, Aberystwyth University, Wales,
UK from an in-house collection. All these strains had demonstrated capability for
metabolising aromatic compounds (David Bryant, Aberystwyth University; personal
communication).
196
4.3.3 MICROBIAL CULTURE DEVELOPMENT AND MAINTENANCE
4.3.3.1 Fungal mycelium development and maintenance
Mycelial agar pieces of 5mm x 5mm from the margin of an actively growing colony from
a stock YPD agar plate were carefully placed approximately 5 mm distance from the edge
of a fresh YPD agar plate. Inoculated plates were sealed with parafilm and placed in an
incubator at 28 oC (adapted from Muthukumar et al., 2011). Inoculum was obtained by
growing the fungus until a densely sporulating mycelial mat covered the entire agar
surface and the plate was stored at 4 oC for further use.
4.3.3.2 Fungal spore preparation and maintenance

Preparation of fungal spores was carried out adapting the method described by Harman
et al., (1991). Spores were removed from the inoculated YPD agar-based media by
scraping with a spatula. These spores were suspended in 10 mL of water by mixing with
a magnetic stirrer, filtered through four layers of sterile cheesecloth to remove hyphae,
and then centrifuged at 10 000 x g for 15 minutes. The pellet was resuspended in 1 mL
of distilled water, removed into sterile 2 mL centrifuge tubes with glycerol to give a final
concentration of 15 %. The suspension was vortexed, aliquoted in 200 μL volumes and
stored at -80 oC for further use.
4.3.3.3 Bacterial culture growth and development

The streak plate method (Soestbergen et al., 1969) was followed for the growth of
bacterial cultures. Following the procedures in the literature (Hinsa and O’Toole, 2006;
Ruiz-manzano et al., 2005) an inoculum suspension of 0.1 mL from a glycerol stock was
pipetted directly onto an LB agar plate and streaked over the surface using a plastic loop.
Inoculated plates were sealed with parafilm, placed in an incubator at 30 oC for 24 hours
and stored at 4 oC for further use. In order to prepare new glycerol stocks, a single colony
was used for inoculation of 10 mL fresh LB media. Following incubation at 30 oC with
shaking (180 rpm) for 24 hours, the culture was mixed with glycerol to final
concentration of 15 %, aliquoted into sterile 2mL centrifuge tubes and stored at -80 oC.
197
4.3.4 MEDIA FOR CULTURING AND MAINTENANCE OF MICROORGANISMS
4.3.4.1 Yeast Peptone Dextrose media (YPD) for fungal growth
Yeast Peptone Dextrose media (YPD) was used for culturing and maintenance of fungal
species. The composition of YPD media is given in Table 4.1 . In order to prepare the
media, 50 g of powdered components shown below were dissolved in 1L of distilled
water and the solution was sterilised by autoclaving at 121 oC for 15 minutes.
Table 4.1: Composition of YPD media.
Compound g L-1
Bacterial peptone 20.0

Yeast extract 10.0
Glucose 20.0
*Agar 15.0
*Added for solid media preparation only, before making up

to 1L and autoclaving.
4.3.4.2 Luria-Bertani media (LB) for bacterial growth

Luria-Bertani media (LB) was used for culturing and maintenance of bacterial species.
Commercial composition of LB media is given in Table 4.2. In order to prepare the media,
25 g of powdered components shown below were dissolved in 1L of distilled water and
the solution was sterilised by autoclaving at 121 oC for 15 minutes.
Table 4.2: Composition of LB media.
Compound g L-1
Tryptone 10.0
NaCl 10.0
Yeast Extract 5.0
*Agar 15.0
*Added for solid media preparation only, before making up

to 1L and autoclaving.
198
4.3.5 MINIMAL MEDIA FOR SCREENING OF MICROORGANISMS AND LIGNINOLYTIC ENZYMES
4.3.5.1 Yeast Nitrogen Base media (YNB)
Yeast Nitrogen Base media (YNB) was used for screening of lignin-metabolising fungi and
ligninolytic enzymes. The composition of YNB media is given in Table 4.3. In order to
prepare 1 x working solution, 100 mL of 10 x stock solution was added to 900 mL of
distilled water and the suspension was sterilised by autoclaving at 121 oC for 15 minutes.
199
Table 4.3: Composition of YNB media.
Compound Amount/L
g L-1
Nitrogen Sources
Ammonium sulfate 5.0
Salts
Potassium phosphate monobasic 1.0
Magnesium sulfate 0.5
Sodium chloride 0.1
Calcium chloride 0.1
ug L -1
Vitamins
Biotin 2.0
Calcium pantothenate 400.0
Folic acid 2.0
Inositol 2,000.0
Nicotinic acid 450.0
p-Aminobenzoic acid 200.0
Pyridoxine HCl 400.0
Riboflavin 200.0
Thiamine HCl 400.0
Citric acid 100.000.0
Trace elements
Boric acid 500.0
Copper sulfate 40.0
Potassium iodide 100.0
Ferric chloride 200.0
Manganese sulfate 400.0
Sodium molybdate 200.0
Zinc sulfate 400.0
g L -1
*Agar 15.0
*Added for solid media preparation only, before making up to 1L

and autoclaving.
200
4.3.5.2 M9 media (minimal salts)
M9 media was used for screening of lignin-metabolising bacteria and ligninolytic
enzymes. Commercial composition of M9 media is given in Table 4.4. In order to prepare
1 x working solution, 200 mL of 5 x stock solution was added to 800 mL of sterile water
and the suspension was sterilised by autoclaving at 121 oC for 15 minutes. The medium
was cooled down to 50 oC and supplemented with 2 mL of 1M MgSO4 and 100 μL of 1M
CaCl2, (filter-sterilized before).
Table 4.4: Composition of M9 media (minimal salts).
Compound g L-1
(Minimal salts)
Sodium phosphate dibasic 33.9
(anhydrous)
Potassium phosphate monobasic 15.0 .
Ammonium chloride 5.0
Sodium chloride 2.5
g L-1
Trace metals
Magnessium sulfate (1M) 120.37
Calcium chloride dihydrate 147.01
g L-1
*Agar 15.0 .
*Added for solid media preparation only, before making up to 1L and

autoclaving
201
4.3.6 SPECIFIC MEDIA FOR PRIMARY SCREENING OF LIGNIN-METABOLISING FUNGI AND BACTERIA
All media were made as described below, using YNB and M9 minimal salts as base media
and different amounts of CIMV lignin as a carbon and energy source.
4.3.6.1 YNB media supplemented with CIMV lignin

YNB solid media (pH 5) and YNB liquid media (pH 5 and pH 7) were used for screening of
lignin-metabolising fungi. These were composed of Yeast Nitrogen Base (YNB) (prepared
as described in section 4.3.5.1) supplemented with CIMV lignin. Freeze-dried CIMV lignin
was added individually to media in the following amounts: 0.25 %, 0.5 %, 1 %, and 2 %
(w/v). NaOH was used to adjust media to pH 7. After addition of agar (1.5 % w/v) (for
solid media only), media was sterilized by autoclaving at 121 oC for 15 minutes and then
cooled down to 50 oC. Media used for controls were prepared as above, without adding
carbon and energy source (negative control) or replacing lignin with 0.5 % (w/v) of
glucose (positive control).
4.3.6.2 M9 minimal media supplemented with CIMV lignin

M9 minimal solid media and M9 minimal liquid media (pH 7) were used for screening of
lignin-metabolising bacteria. These were composed of M9 (minimal salts) (prepared as
described in section 4.3.5.2) supplemented with CIMV lignin. Freeze-dried CIMV lignin
was added to the media at 0.25 % (w/v). After addition of agar at 1.5 % (w/v) (for solid
media only), media was sterilized by autoclaving at 121 oC for 15 minutes, cooled down
to 50 oC and supplemented with filter-sterilized Trace Element Solution (Table 4.4).
Media used for controls were prepared as above, either without addition of a carbon
and energy source (negative control) or replacing lignin with 0.25 % (w/v) glucose
(positive control).
4.3.7 SPECIFIC MEDIA FOR PRIMARY SCREENING OF LIGNINOLYTIC ENZYMES
All media were made as described below, using YNB and M9 minimal salts as a base and
different amounts of CIMV lignin, glucose and methylene blue as a carbon and energy
source. Methylene blue solution was also used as a dye indicator.
202
4.3.7.1 YNB solid media supplemented with lignin and methylene blue
Two sets of YNB solid media at pH 5 and pH 7 were used to screen for ligninolytic
enzymes and were composed of Yeast Nitrogen Base (YNB) (prepared as described in
section 4.3.5.1) supplemented with either CIMV lignin and methylene blue solution or
methylene blue solution alone. CIMV lignin was freeze-dried and added individually to
media in the following amounts: 0.25 %, 0.5 %, 1 % and 2 % (w/v). NaOH was used to
adjust media to pH 7. After addition of 1.5 % (w/v) agar, the media was sterilized at 121
oC for 15 minutes, cooled down to 50 oC and supplemented with 5mL of methylene blue
solution from a filter-sterilised stock solution (10 mg/mL in 70 % methanol) to give a final
concentration of 0.05 g/L. Media supplemented with methylene blue only were
prepared as above, without adding any extra carbon and energy source.
4.3.7.2 M9 solid media supplemented with CIMV lignin and methylene blue
M9 solid media at pH7 was used for screening of ligninolytic enzymes and was composed
of M9 (minimal salts) (prepared as described in section 4.3.5.2) supplemented with either
CIMV lignin and methylene blue solution, glucose and methylene blue solution or
methylene blue solution alone. 0.25 % (w/v) of freeze-dried CIMV lignin/ or 0.25 % (w/v)
of glucose was added to the media. The solid media was prepared as above using the
minimal M9 media.
4.3.8 CHARACTERISATION OF LIGNIN METABOLISING FUNGI AND BACTERIA
All experiments were conducted as described below. The conditions for preliminary
growth experiments (such as pH and temperature) were determined based on a
literature search. The fungal species, T. harzianum was grown at pH 5 and pH 7 and
incubated at 28 oC as earlier reported by Reetha et al., (2014). Additionally, Pelomonas
saccharophila and Pseudomonas species were grown at pH 7 and 30 oC as reported by
Munna et al (2015) and Espeso et al., (2016). Growth was determined at time intervals Field Code Changed
of 5 and/or 10 days, depending on the type of media and ligninolytic capability of the
microorganisms. There were two control experiments. For the positive control,
Misanthus giganteus and lignin incubated with T. harzianum was replaced by 0.5 % (w/v)
glucose. Additionally, lignin incubated with Pelomonas saccharophila and Pseudomonas
species was replaced by 0.25 % (w/v) of glucose. These concentrations were selected for
203
practical reasons to equate with lignin supplement of 0.25 %-0.5 % (w/v). Inoculum Commented [AW[102]: Need to add sentence here to explain
growth in media without carbon and energy source was used as the negative control.
4.3.8.1 Growth of T. harzianum in YNB liquid media supplemented with Miscanthus

giganteus
All fungal cultures were grown in 250-mL shake flasks with 100 mL of YNB minimal media
(prepared as described in section 4.3.5.1) supplemented with Miscanthus giganteus (MG)
as the sole carbon and energy source. For this preliminary trial, the experiment was
carried out only at pH 5, based on the protocols of Rubeena et al., (2013). These authors
screened for lignocellulolytic activities of a T. harzianum strain. Prior to cultivation, MG
was freeze-dried, cut to small pieces, milled (if applicable), autoclaved and added to
duplicate sets of sterile media in the following amounts: 25 % (w/v) (milled <2mm), 25
% (w/v) (unmilled), 10 % (w/v) (unmilled) and 5 % (w/v) (unmilled). Each flask with the
appropriate amount of biomass in the media, was individually inoculated with 100 μL of
the glycerol-spores suspension (50-fold dilution) and/or mycelial agar pieces of 5mm x
5mm (prepared as described in section 4.3.3). Cultures were grown in a rotary shaker
with shaking at 180 rpm at 28 oC for 10 days. The growth was monitored at time intervals
of 5 and 10 days.
4.3.8.2 Growth of T. harzianum on YNB solid media supplemented with CIMV lignin
Mycelial agar pieces of 5mm x 5mm (prepared as described in section 4.3.3) were
inoculated onto YNB media agar plates supplemented with 0.25 %, 0.5 % and 1 % (w/v)
of lignin (pH 5) (prepared as described in section 4.3.6.1). This study was carried out only
at pH 5, for reasons discussed above. Inoculated plates were incubated at 28 oC for 5
days and photographed.
4.3.8.3 Growth of T. harzianum in YNB liquid media supplemented with CIMV lignin
All cultures were grown in 250-mL shake flasks in 100 mL YNB liquid media supplemented
with 0.25 %, 0.5 % , 1 % and 2 % (w/v) of CIMV lignin (pH 5 and pH 7) (prepared as
described in section 4.3.6.1). An additional treatment at pH 7 was included, because
maximum growth was observed at this pH in previous studies (Reetha et al., 2014) Each
flask with the appropriate amount of lignin in media, was inoculated with 100 μL of the
glycerol-spores suspension (50-fold dilution) and mycelial agar pieces of 5m x 5mm
(prepared as described in section 4.3.3). Cultures were mixed thoroughly, by keeping the
204
flasks on a rotary shaker at 180 rpm and 28 oC for 10 days. The growth was determined,
at time intervals of 5 and 10 days.
4.3.8.4 Growth of P. putida, P. saccharophila, P. fluorescens on M9 solid media

supplemented with CIMV lignin
Prior to growth, pure colonies from original LB solid media (prepared as described in
section 4.3.3) were inoculated in 10 mL of fresh LB liquid media and incubated at 30 oC
with 180 rpm for 24 hours. A 100 μL of inoculum of each strain was spread over M9 solid
media plates supplemented with either 0.25 % (w/v) of lignin or 0.25 % (w/v) of lignin
and 0.25 % (w/v) of glucose (pH 7) (prepared as described in section 4.3.6.2), incubated
at 30 oC for 10 days and photographed.
4.3.8.5 Growth of P. putida, P. saccharophila, P. fluorescens in M9 liquid media with CIMV

lignin
All cultures were grown in 250 mL shake flasks, filled with 100 mL of M9 liquid media
supplemented with 0.25 % (w/v) of CIMV lignin (pH 7) (prepared as described in section
4.3.6.2). Prior to growth, pure colonies from original LB solid media (prepared as
described in section 4.3.3) were inoculated in 10 mL fresh LB liquid media and incubated
at 30 oC with shaking at 180 rpm for 24 hours. Each flask filled with M9 liquid media
supplemented with lignin, was individually inoculated with 2 mL of the inoculum of each
strain. Cultures were thoroughly mixed and 2mL of suspension was transferred
immediately to 100 mL fresh media to ensure that observed growth could not be a result
of LB media carried over. All the flasks were incubated at 30 oC for 96 hours, with
constant agitation at 180 rpm and growth was monitored at times interval of 0 and 96
hours. In order to monitor growth in liquid media, 100 μL of fermentation culture from
each flask at 0 hours and 96 hours (10.000-fold dilution) was spread on the surface of
LB agar, sealed with parafilm, incubated at 30 oC for 48 hours and photographed. Commented [AW[103]: Mention if you counted colonies
4.3.9 ENZYME SCREENING ASSAYS
All enzyme assays were conducted as described below. Methods for assessing ligninolytic
activity both: from fungal and bacterial strains were developed referring to procedures
described by Bandounas et al., (2011), Bholay et al., (2012), Sasikumar et al., (2014), Tian
et al., (2016) and Ghribi et al., (2016). Enzyme activities were assessed at pH 5 and pH
205
7, taking into account reported optimal pH for fungal and bacterial ligninolytic activities.
Laccase from T. versicolor was chosen from a range of oxidative enzymes and used for
positive control in dye decolourisation and laccase activity assays. The optimal
concentration was chosen based on a series of 5 different dilutions carried out prior to
experiments.
4.3.9.1 Dye-decolourisation assay on solid media

Fungal and bacterial strains were screened based on their ability to form clear zones
around fungal or bacterial growth on solid media supplemented with different amounts
of lignin, glucose and methylene blue. Decolourisation was determined after 5 or/and 10
days, depending on visual observations. The positive control was set up by using 100 μL
of 0.025 mg/mL laccase enzyme solution from Trametetes versiciolor instead of Commented [AW[104]: Should put units of activity here-
Noelia may be able to give this info
inoculum. Uninoculated plates were used as negative control for spontaneous dye
decolourisation.
4.3.9.1.1 Ligninolytic activity of enzymes from T. harzianum on YNB solid media
Mycelial agar pieces of 5mm x 5mm (prepared as described in section 4.3.3) were placed
onto YNB solid media supplemented with either methylene blue (0.05 g/L) alone or 0.25
% , 0.5 %, 1 % (w/v) of lignin and methylene blue (0.05 g/L) (pH 5 and pH 7) (prepared as
described in section 4.3.7.1). Inoculated plates were incubated at 28 oC for 5 days and
photographed.
4.3.9.1.2 Ligninolytic activity of enzymes from P. putida, P. saccharophila and P.

fluorescens on M9 solid media
Prior to the enzyme assay, single colonies from an original culture LB plate (prepared as
described in section 4.3.3) were inoculated into 10 mL fresh LB liquid medium and grown
for 24 hours at 30 oC with 180 rpm. A 100 μL cultures of each strain were spread and
streaked onto M9 solid media supplemented with either methylene blue (0.05 g/L)
alone, methylene blue (0.05 g/L) and 0.25 % (w/v) of lignin or methylene blue (0.05 g/L)
and 0.25 % (w/v) of glucose (pH 7) (prepared as described in section 4.3.7.2). The
inoculated plates were incubated at 30 oC for 10 days and growth was photographed.
206
4.3.9.2 Dye-decolourisation assay in liquid media
Fungal and bacterial strains were screened based on their ability to decolourise dye in
fermentation broth supplemented with lignin. For the positive control, the crude enzyme
extract was substituted with 100 μL of 0.025 mg/mL laccase enzyme solution from Commented [AW[105]: Add units if possible
Trametetes versicolor. For the negative control, crude enzyme extract was replaced with
buffer and/ or cell-free supernatant.
4.3.9.2.1 Ligninolytic activity of enzymes from T. harzianum grown in YNB liquid media
The activity of enzymes from T. harzianum fermentation broth supplemented with lignin
was assessed following 10 days incubation at pH 5 and pH 7. The culture was centrifuged
at 8 000 rpm for 10 minutes at 4 oC, and the supernatant was used for enzyme assay.
The assay reactions were set up in 2 mL centrifuge tubes containing 900 μL of master
mix including: McIlvaine buffer (0.2M disosodium hydrogen phosphate, 0.1M citric acid)
(McIlvaine, 1921), methylene blue (0.0 5g/L) and 100 μL of spent culture supernatant to
make up to 1mL. 900 μL of master mix solution was mixed with 100 μL of spent culture
supernatant, incubated for 10 minutes at room temperature and photographed.
4.3.10 LACCASE ACTIVITY ASSAY

4.3.10.1 Laccase activity of extracellular enzymes produced by T. harzianum
Extracellular enzymes produced by T. harzianum following 10 days incubation in YNB
media supplemented with MG (as described in section 4.3.8.1), were assayed for laccase
activity at pH 5 and pH 7. The assay reaction was performed in 2 mL centrifuge tube
containing 450 μL of master mix including McIlvaine buffer (0.2M disodium hydrogen
phosphate, 0.1M citric acid) (McIlvaine, 1921), 1mM ABTS solution and 50 μL of enzyme
extract to make up to 500 μL. A 450 μL aliquot of the master mix solution was added to
50 μL enzyme extract, incubated for 30 minutes at 30 oC and photographed. For the
positive control, enzyme extract was replaced with 50 μL 0.025 mg/mL of laccase from
Trametetes versiciolor and buffer replaced the enzyme extract in the negative control.
207
4.3.11 PROTEIN PURIFICATION
4.3.11.1 Ammonium sulphate precipitation for extracellular enzymes
Prior to processing, fermentation broth from each flask was vacuum-filtered using a
paper filter (Whatman No. 1) to separate the broth from lignin. The flow through was
transferred into 50 mL of centrifuge tubes and centrifuged at 8500 rpm for 15 minutes
at 4 oC. The supernatant was transferred into a conical flask (placed on ice), and
precipitated slowly to give final saturation level of 90 % of Ammonium sulphate, with
constant mixing to obtain optimum precipitation of proteins. Following 30 minutes
incubation, the mixture was centrifuged at 8500 rpm for 15 minutes at 4 oC to pellet the
protein. The supernatant was discarded, residual pellet was re-suspended in 0.5 mL 50
mM Tris-HCl buffer, pH 7 with protease inhibitors (1000 units/mL Trasylol, 0.2M
Phenylmethylsulfonylfluoride (PMSF), 0.1M 1,10 phenantroline, 0.1M benzamidine)
vortexed well and retained for the desalting process.
4.3.11.2 Desalting proteins

Protein desalting was carried out according to the protocol provided by Dr Ana Winters
(Aberystwyth University, IBERS). Bio-Gel P Polycramide Gel (6 g) from Bio-Rad (Hercules,
CA, USA) was suspended in 200 mL of distilled water, mixed well and allowed to swell
overnight. Distilled water was decanted and the gel was washed thrice with distilled
water. To prepare the gel for desalting the protein sample, it was suspended in 100 mL
of 50mM Tris-HCl buffer at pH 7, mixed, allowed to settle and the buffer was decanted.
This was followed by two washes of 100 mL 50mM Tris-HCl buffer with proteases at pH
7 (prepared as described in section 4.3.11.1), mixed, allowed to settle and finally partly
decanted to produce a gel ready to be loaded into desalting columns. The desalting
columns were prepared by cutting out round discs from micro-cloth and placing them at
the bottom of 10 mL syringe barrels to prevent the gel from escaping through the outlet.
On the top of each disc, 10 mL of previously agitated gel suspension was added slowly to
the syringe taking care not to disturb discs at the bottom of syringe barrels. Syringe
barrels containing gel suspension were placed inside a 50 mL centrifuge tube and
centrifuged at 1200 x g for 6 minutes. The liquid at the bottom of the centrifuge tube
was discarded and the tube dried out with a paper towel. A 0.5 mL volume of crude
extracellular extract was loaded on the top of the gel in each of desalting columns and
208
centrifuged again at 1200 x g for 6 minutes. Following centrifugation, each desalted
protein solution was collected in a centrifuge tube, aliquoted, transferred into a 2mL
microcentrifuge tube and stored at -80 oC for further assays.
4.4 RESULTS
4.4.1 PRIMARY SCREENING OF T. HARZIANUM FOR GROWTH ON A LIGNOCELLULOSIC FEEDSTOCK AND
EXPRESSION OF LIGNINOLYTIC ENZYMES CAPABLE OF METABOLISING LIGNIN
Primary screening was carried out to establish the growth of T. harzianum on

lignocellulosic feedstock and production of ligninolytic enzymes.
4.4.1.1 Screening T. harzianum for growth in YNB liquid media supplemented with
Miscanthus giganteus
T. harzianum spore suspension and fungal mycelia was inoculated into YNB liquid media
supplemented with Miscanthus giganteus as the sole carbon and energy source and
incubated at pH 5 and 28 oC for 10 days (as described in section 4.3.8.1). The results of
the screening showed, that T. harzianum was capable of utilizing lignocellulosic material
as a carbon and energy source for its growth (Table 4.5). As the rate of growth of fungal
mycelia and spores was very slow in media supplemented with 5 % (w/v) of Miscanthus
giganteus, cultures were assessed after 5 and 10 days incubation. However, there was
no variation observed between both time intervals. With respect to different
concentrations of feedstock, good growth was observed in media supplemented with 25
% (w/v) of unmilled MG, while growth on the same amount of milled feedstock was
rather moderate. The presence of 5 % (w/v) and 10 % (w/v) of MG, resulted in poor
growth, mostly visualized as a biofilm formation on the edges of the flasks (Appendix,
Figures 1 and 2). Following positive results of screening, the ligninolytic capability of T.
harzianum was investigated further.
209
Table 4.5: Growth of T. harzianum in YNB liquid media supplemented with different amounts of
Miscanthus giganteus (MG) on a weight per volume basis (w/v), at pH 5 and 28 oC. Control – : YNB liquid
media and inoculum, Control +: YNB liquid media with 0.5 % (w/v) of glucose and inoculum.
Fungal tissue Extent of growth
Media
25% 25% 10% 5% C- C+ 25 % 25% 10% C- C+

5% Commented [AW[106]: Delete w/v/ move 5 days incubation
MG MG MG MG MG MG MG
MG and 10 days incubation above, delete space between % and number
(ML) (NM) (NM) (NM) (ML) (NM) (NM)
(NM)
5 days 10 days
++ +++ + + - GD ++ +++ + + - GD
T.harzianum
spores
++ +++ + + - GD ++ +++ + + - GD
T. harzianum
mycelium
(ML) = milled, (NM) = not milled, +++ = good growth, ++ = moderate growth, + = poor growth, - = no growth,
GD – growth detected
4.4.1.2 Preparation and screening for extracellular laccase activity in spent culture media
Extracellular enzymes produced by T. harzianum in YNB liquid media supplemented with
different amounts of Miscanthus giganteus (MG) were precipitated from culture media
after 10 days of incubation and screened for laccase activity with ABTS (as described in
sections 4.3.11 and 4.3.10. Results of the assay were determined visually in an assay
mixture at pH 5 and pH 7, to test the effect of pH on enzyme expression, and are shown
in Table 4.6. The appearance of a green colour at pH 5, indicated positive activity of
enzyme precipitated from YNB media, and was only observed in the case of media
supplemented with 25 % (w/v) of unmilled MG (Appendix, Figure 3). There was no laccase
activity detected at pH 7.
210
Table 4.6: Laccase activity of extracellular enzymes produced by T. harzianum in YNB liquid media with
different amounts of Miscanthus giganteus (MG) on a weight per volume basis (w/v), determined after
30 minutes at pH 5, pH 7 and 30 oC. Control-: no enzyme, Control + laccase from T. versicolor.
Fungal tissue Laccase activity
Media
25% 25% 10% 5% C- C+ 25% 25% 10% 5% C- C+ Commented [AW[107]: As above

MG MG MG MG MG MG MG MG
(ML) (NM) (NM) (NM) (ML) (NM) (NM) (NM)
pH 5 pH 7
T.harzianum - + - - - + - - - - - +
spores
T. harzianum - + - - - + - - - - - +
mycelium
+ = positive for laccase activity, - = negative for laccase activity
4.4.2 PRIMARY SCREENING OF T. HARZIANUM FOR GROWTH ON ORGANOSOLV LIGNIN AND EXPRESSION OF
LIGNINOLYTIC ENZYMES CAPABLE OF METABOLISING LIGNIN
Based on results in section 4.4.1, showing ligninolytic capability of T. harzianum when

grown with lignocellulosic feedstock as the C-source, the strain was further tested for
the ability to grow and produce ligninolytic enzymes in minimal media supplemented
with organosolv lignin as the sole carbon and energy source. For method development,
all work was carried out using commercial CIMV organosolv lignin. Due to problems with
setting minimal solid media, the screening test and assay for ligninolytic activity could
not be performed in media supplemented with > 1 % (w/v) of CIMV lignin.
4.4.2.1 Screening T. harzianum for growth on YNB solid media supplemented with CIMV
lignin
T. harzianum spore suspension and mycelia were inoculated onto YNB solid media
supplemented with CIMV lignin as the sole carbon and energy sources to verify that the
211
strain can grow on lignin polymer at pH 5 and 28 oC (as described in section 4.3.8.2). The
results of the screening showed, that T. harzianum was capable of utilizing lignin as a
carbon and energy source for its growth in screening media supplemented with: 0.25 %
,0.5 % and 1 % (w/v) of lignin. In order to confirm, that growth was a result of lignin
utilisation and not media carried over with mycelia, an experiment was conducted by
inoculating T. harzianum from YNB lignin-based media onto a fresh plate. Due to the dark
brown colour of the media, the intensity of growth was difficult to evaluate. Examples of
T. harzianum growth on solid media supplemented with different amount of CIMV lignin
are shown in Figure 4.1. Following positive results of the screening, ligninolytic capability
of T. harzianum was investigated further.
Figure 4.1: Growth of T. harzianum on YNB solid media supplemented with different amount of CIMV
lignin after 5 days incubation at pH 5 and 28 oC. Test: A) YNB + lignin 0.25 % (w/v) + agar, B) YNB + lignin
0.5 % (w/v) + agar, C) YNB + lignin 1 % (w/v) + agar, Control -: YNB + agar + inoculum, Control +: YNB +
glucose 0.5 % (w/v) + agar + inoculum.
4.4.2.2 Ligninolytic enzyme activity detection on YNB solid media

T. harzianum was inoculated on YNB agar plates supplemented with either methylene
blue alone or methylene blue and CIMV lignin as a carbon and energy source, and
screened for the ability to grow and form clear zones at pH 5 and pH 7 when incubated
at 28 oC for 5 days (as described in section 4.3.9.1.1). Dye decolourisation, observed due
to complete methylene blue oxidation indicated ligninolytic activity of the fungal strain
screened for this study. The results of the assay are shown in Table 4.7. The plates
showed lack of clearance zones around T. harzianum grown on YNB agar plates
supplemented with methylene blue as the sole carbon and energy source. However, T.
harzianum growth and decolourisation zones were observed on all three screening
media supplemented with different amounts of lignin: 0.25 % , 0.5 %, and 1 % (w/v), at
pH 5 and pH 7. The activity with dye was visualised within 3 days of incubation and was
212
most efficient in media supplemented with 0.25 % (w/v) of lignin, both at pH 5 and pH 7.
The decolourisation process in media supplemented with 0.5 % (w/v) of lignin, was much
higher at pH 5. There were no evident differences of dye degradation in media
supplemented with 1 % (w/v) of lignin. However, due to lack of homogenous distribution
across the plate final conclusions could not be made (Appendix, Figures 4 and 5).
Following the positive results in screening for ligninolytic activity on YNB solid media
supplemented with lignin and lignin-like dye, T. harzianum was subjected to further
growth experiment in YNB liquid media.
213
Table 4.7: Dye decolourisation during growth of T. harzianum on YNB solid media supplemented with
methylene blue (MB) and different amounts of lignin on a weight per volume basis (w/v), at pH 5 and pH
7 incubated at 28 oC for 5 days. Control-: uninoculated plate, Control +: plate supplemented with laccase
from T. versicolor.
Fungal tissue Dye decolourisation
Medium
MB MB MB MB MB MB MB MB
0.25% 0.5% 1% 0.25% 0.5% 1%
lignin lignin lignin lignin lignin lignin
pH 5 pH 7
T.harzianum
mycelium - +++ ++ ND - +++ ++ ND
C- - - - - - - - -
C+ ++ ++ ++ ++ ++ ++ ++ ++
+++ = intense decolourisation, ++ =moderate decolourisation, + = faint decolourisation, - = no decolourisation

ND= not possible to determine
4.4.2.3 Screening T. harzianum for growth in YNB liquid media supplemented with CIMV
lignin
T. harzianum was inoculated into YNB liquid media supplemented with CIMV lignin as
the sole carbon and energy source to monitor if the strain can grow on lignin polymer at
pH 5 or pH 7 at 28 oC for 10 days (as described in section 4.3.8.3). Growth was monitored
after 5 and 10 days of incubation. However, due to the dark brown colour of broth, there
was no clear evidence of growth in flasks inoculated with fungal mycelium/spores at both
time intervals and the final conclusions could not be determined (Tables 4.8 and 4.9). The
ligninolytic capability of T. harzianum in liquid culture was further investigated by
carrying out an assay for oxidative enzyme activity.
214
Table 4.8: Growth of T. harzianum after 10 days growth in YNB liquid media supplemented with different
amounts of lignin on a weight per volume basis (w/v), at pH 5 and 28 oC. Control -: YNB liquid media and
inoculum, Control +: YNB liquid media with 0.5 % (w/v) of glucose and inoculum.
Growth
Fungal tissue
Medium
0.25% 0.5% 1% 2% C- C+ 0.25% 0.5% 1% 2% C- C+

lignin lignin lignin lignin lignin lignin lignin lignin Commented [AW[108]: Delete w/v here
5 days 10 days
T.harzianum ND ND ND ND - + ND ND ND ND - +
spores
T. harzianum ND ND ND ND - + ND ND ND ND - +
mycelium
ND = not detected + = growth, - = no growth
215
Table 4.9: Growth of T. harzianum in YNB liquid media supplemented with different amounts of lignin on
a weight per volume basis (w/v) at pH 7 incubated at 28 oC for 10 days. Control -: YNB liquid media and
inoculum, Control +: YNB liquid media with 0.5 % (w/v) of glucose and inoculum.
Growth
Fungal tissue
Media
0.25% 0.5% 1% 2% C- C+ 0.25% 0.5% 1% C- C+

2%
lignin lignin lignin lignin lignin lignin lignin
lignin
5 days 10 days
T.harzianum
ND ND ND ND - + ND ND ND ND - +
spores
T. harzianum
ND ND ND ND - + ND ND ND ND - +
mycelium
ND = not detected, + = growth, - = no growth
4.4.2.4 Ligninolytic enzyme activity assay in YNB liquid media

In order to evaluate ligninolytic capability of T. harzianum in fermentation broth, the
liquid culture from section 4.4.2.3 was centrifuged and further subjected to ligninolytic
activity assay for the presence of extracellular enzymes (as described in section 4.3.9.2.1).
Results of the assay were determined visually at pH 5 and pH 7 (to test the effect of pH
on enzymes expression) and are shown in Table 4.10. Decolourisation of methylene blue
was observed in all spent culture supernatants at pH 5, and clearly indicated ligninolytic
activity of the studied fungi. The highest decolourisation was observed with spent culture
supernatants supplemented with 0.25 % and 0.5 % (w/v) of lignin. In turn, the lowest
decolourisation was detected in spent culture supernatants supplemented with 2 %
(w/v) of lignin. There was no evidence of ligninolytic activity at pH 7 (Appendix, Figures 6
and 7).
216
Table 4.10: Dye decolourisation in spent culture supernatants after incubation of T. harzianum in YNB
liquid media with different amounts of lignin on a weight per volume (w/v) basis at pH 5 or pH 7, and
incubated at 28 oC for 10 days. Control-: no enzyme, Control +: laccase from T. versicolor.
Dye decolourisation
Fungal tissue
Media
0.25% 0.5% 1% 2% C- C+ 0.25% 0.5% 1% C- C+

2%
lignin lignin lignin lignin lignin lignin lignin
lignin
pH 5 pH 7
+++ +++ ++ + - DD - - - - - DD
T.harzianum
spores
T. harzianum
+++ +++ ++ + - DD - - - - - DD
mycelium
+++ = intense decolourisation, ++ =moderate decolourisation, + = faint decolourisation, - = no decolourisation
DD = decolourisation detected
4.4.3 PRIMARY SCREENING OF BACTERIAL STRAINS FOR GROWTH ON ORGANOSOLV LIGNIN AND EXPRESSION
OF LIGNINOLYTIC ENZYMES CAPABLE OF METABOLISING LIGNIN
Following the results in section 4.4.2, showing ligninolytic capability of T.harzianum in

the presence of CIMV lignin, similar screening tests were carried out for bacterial strains.
All solid media were supplemented with 0.25 % lignin only. This was chosen for practical
reasons, such as ease of media preparation and growth estimation.
4.4.3.1 Screening P. putida, P. saccharophila, P. fluorescens for growth on M9 solid media

P.putida, P.saccharophila and P. fluorescens were inoculated onto M9 solid media
supplemented with CIMV lignin as the sole carbon and energy source to test for growth
of the strain on organosolv lignin at pH 7 and 30 oC for 10 days (as described in section
4.3.8.4). As no growth was observed after 5 days, the plates were incubated for a further
217
5 days. However, the final results of the screening showed, that none of the strains were
able to utilize lignin as a carbon and energy source for growth on solid minimal media
(Table 4.11/Appendix, Figure 8). Following these negative results, ligninolytic capability
of bacteria on solid minimal media environment was further investigated by carrying out
enzyme activity assay.
Table 4.11: Growth of P. putida, P. saccharophila and P. fluorescens after 10 days incubation on M9 solid
media supplemented with 0.25 % (w/v ) of lignin at pH 7 and 30 oC. Control -: M9 solid media and
inoculum, Control +: M9 solid media with 0.25 % (w/v) of glucose and inoculum.
Bacterial strain Growth
Media
0.25% C- C+
lignin
- - +
P.putida
P.saccharophila
- - +
P.fluorescens - - +
+ = growth, - = no growth
4.4.3.2 Ligninolytic enzyme activity assay on M9 solid media

P.putida, P.saccharophila and P. fluorescens were inoculated onto M9 agar plate
supplemented with either methylene blue alone, methylene blue and glucose or
methylene blue and CIMV lignin as the carbon and energy source, and screened for the
ability to grow and form clearance zones at pH 7 and 30 oC for 10 days (as described in
section 4.3.9.1.2). As no decolourisation was observed following the first 5 days
incubation and a further 5 days, these results indicated, that none of the microorganisms
were able to produce clearance zones on all three screening media due to a lack of
ligninolytic activity (Table 4.12). However, P. putida and P. fluorescens showed clear
218
growth on plates with M9 minimal media, methylene blue and glucose within 3 days of
incubation (Appendix, Figures 9, 10 and 11). These results confirmed the findings of the
previous experiment that these strains could not utilise lignin as a carbon and energy
source in these growth conditions.
Table 4.12: Dye decolourisation during growth of P. putida, P. saccharophila, P. fluorescens on M9 solid
media supplemented with methylene blue, glucose and lignin on a weight per volume basis. for 10 days
at pH 7 and 30 oC.Control-: uninoculated plate, Control +: plate supplemented with laccase from T.
versicolor.
Bacterial strain Dye decolourisation
Media
MB MB C+ C-
MB
0.25% 0.25%
glucose lignn
P. putida
- - + -
-
P. saccharophila
- - - + -
P. fluorescens
- - - + -
+ = decolourisation, - = no decolourisation
4.4.3.3 Screening P. putida, P. saccharophila, P. fluorescens for growth in M9 liquid media

Based on the results presented in section 4.4.3.2, showing lack of ligninolytic capability
of bacterial strains on solid media, P.putida, P.saccharophila and P. fluorescens were
inoculated into M9 liquid media supplemented with CIMV lignin as the sole carbon and
energy source (as described in section 4.3.8.5) and incubated for 96 hours. Cultures were
screened for active growth at 0 and 96 hours by spreading 100 μL liquid culture onto LB
plates and incubating for 48 hours (Figure 4.2). Results of the screening showed, that all
219
3 strains were able to utilize lignin as a carbon and energy source for growth by 96 hours
of incubation.
Figure 4.2: Growth of P.putida (1), P. saccharophila (2), P. fluorescens (3) plated on LB plates after
incubation in M9 liquid media supplemented with 0.25 % (w/v) of lignin for 96 hours at pH 7 and 30 oC
(growth detected after 48 hours). Commented [AW[109]: check labelling
220
4.5 DISCUSSION
There are a wide variety of organisms in the biosphere involved in lignin degradation
including fungi and bacteria (Bholay et al., 2012). However, Aarti et al., (2015)
concluded, that processes for lignin degradation using isolated ligninolytic microbes are
not currently suitable for application at industrial scale. Moreover, taking into account
the potential role of lignin-degrading enzymes in the development of green processes
for biorefineries, the quest for novel biocatalysts with high robustness and outstanding
catalytic performances is currently of utmost importance (Picart et al., 2016).
A common approach in this respect is the enrichment of microbial cultures with lignin as
the sole carbon source and subsequent isolation of single microbial strains that are able
to grow on lignin. Currently, the recognition and knowledge surrounding fungal lignin
degradation is the basis for the most part on ligninolytic research (Fisher and Fong, Field Code Changed
2014). In this study, T. harzianum (white- rot filamentous fungus,) was examined in detail
for the ability to grow on different forms of lignin and produce ligninolytic enzymes. Prior
to screening, optimum conditions were determined for the detection of lignin utilisation.
Temperature, pH, carbon and nitrogen sources are one of the most influencial factors
affecting fungal growth (Aarti et al., 2015). Based on the literature, T. harzianum was
grown at 28 oC and at two different pH (5 and 7); as minimal and maximal growth,
respectively was observed under these conditions in a previous study (Reetha et al., Commented [AW[110]: check not media specific
2014).
The first trials were focused on screening of the selected fungal strain for the ability to
grow on lignin-containing feedstock. The results of the screening, following incubation
for 10 days at pH 5, showed that T. harzianum was able to utilize lignocellulosic material
as a carbon and energy source for its growth in these conditions. The fungal strain
exhibited the best growth in YNB liquid media supplemented with 25 % (w/v) of unmilled
feedstock. In turn, the presence of 10 % (w/v) and 5% (w/v) of milled biomass, resulted
in very poor growth (Table 4.5). 5 % and 10 % levels of Miscanthus giganteus
supplementation resulted in a largely liquid environment while with 25 % of Miscanthus
giganteus, there was a large volume of solids above the liquid level (Appendix, Figures 1
221
and 2). This indicated, that the solid-based environment and the bigger surface
accessibility (as seen with 25 % (w/v) of Miscanthus giganteus) was much more
preferable for this strain. There is evidence to suggest, that these findings are highly
related to morphological and physiological characteristics of Trichoderma species.
According to the literature, these fungi commonly grow from the roots out into the
surrounding soil, forming an external hyphal network which increases uptake of mineral
nutrients (Green et al., 1999).
The capacity to grow on lignocellulosic substrates is directly related to the production of

a broad spectrum of enzymes that act synergistically to deconstruct the plant cell wall
(Bandounas et al., 2011). Extracellular enzymes produced by T. harzianum were
precipitated from the culture media and screened for laccase activity. This activity was
investigated as laccases form one of the main classes of ligninolytic enzymes produced
by Trichoderma species (Gochev and Krastanov, 2007; Hölker et al., 2002). The enzyme
activity was assessed at pH 5 and pH 7 as this spans the growth range for this species.
The appearance of a green colour at pH 5 in assay mixtures indicated positive activity of
enzyme precipitated from YNB media, supplemented with 25 % (w/v) unmilled MG
(Table 4.6) (Appendix, Figure 3). These findings corroborated the outcomes from the
growth screening test described in section 4.4.1.1,where maximal growth was observed
with 25 % (w/v) MG supplemented media. It is worth mentioning, that these results were Commented [AW[111]: Not sure what you mean here
Commented [AS[112R111]: We clarified that
in good agreement with data published in the literature. Abd El Monssef et al., (2016)
and Chakroun et al., (2010) observed, that the optimum pH value for laccase activity
from Trichoderma species ranges between 4-5.5.5. In their research, laccase showed
practically no activity at pH values higher than 6. It is still not clear, whether the laccase
activity is induced so the fungus can utilise lignin as a carbon and energy source or to
assist in cell wall breakdown to make extra carbon and energy sources more accessible.
Undoubtedly, more research would be necessary to understand this phenomenon.
Based on the findings suggesting ligninolytic capability of T. harzianum in the presence

of lignin-containing feedstock, the strain was screened on solid minimal media
supplemented with different amounts of organosolv lignin. For the purpose of method
development, commercial CIMV lignin was chosen. The results of the screening
following 5 days incubation at pH 5 showed, that T. harzianum was capable of utilizing
222
CIMV lignin as the sole carbon and energy source for growth on all tested media,
supplemented with: 0.25 %, 0.5 % and 1 % (w/v) lignin (Figure 4.1). In order to confirm
the ligninolytic capability of the strain investigated this study, mycelia were inoculated
onto YNB agar plates supplemented with either methylene blue or methylene blue and
CIMV lignin as a carbon and energy sources and screened for the ability to grow and form
clearance zones. The capacity of degrading synthetic lignin-mimicking dyes has
frequently been used in assays of oxidative enzymes involved in ligninolytic activity (Tian
et al., 2016). Due to the its close similarity to lignin, methylene blue was used as a dye
indicator of activity (Ghribi et al., 2016). As above, the enzyme activity assay was
assessed at pH 5 and pH 7 following incubation at 28 oC. The results of the analysis
revealed the lack of clearance zones around T. harzianum grown on YNB agar plates
supplemented with methylene blue as the sole carbon and energy source. However, the
fungal strain investigated in this study grew and decolourized all screening media
supplemented with different amounts of lignin: 0.25 %, 0.5 % and 1 % (w/v), at pH 5 and
pH 7 (Table 4.7) (Appendix, Figures 4 and 5). Absence of growth on media supplemented
with methylene blue alone showed, that observed growth was an effect of lignin/lignin-
like dye degradation, rather than a media carried over from original plate. There are Commented [AW[113]: Not clear what you mean here
Commented [AS[114R113]: We clarified that
several reasons which could explain above findings. According to Tian et al., (2016),
thiazine dyes such as methylene blue can only be significantly degraded, when the
concentration of oxidative enzymes reaches a specific level and that is only possible in a
rich media but not in a minimal media. It is very likely, that the thiazine dyes in minimal
media may be degraded by co-metabolism eg. methylene blue and lignin. The low
concentration of dye (0.05 g/L) added to media in this study could suggest, that
methylene blue is not used as a sole carbon and energy source and extra carbon source
is needed as an inducer. The observation of growth and enzyme production in media Commented [AW[115]: check
supplemented with lignin, may support this hypothesis. Similar finding were reported in
the literature. Sekar et al., (2013) showed, that fungal strains including T. viride were not
able to degrade methylene blue in minimal media at low concentration (<0.05 g/L).
However, Jayasinghe et al., (2008) and Taylor et al., (2017) reported some white-rot fungi
species that showed break down of the dye in the presence of rich media such as potato
dextrose agar and malt extract agar. Out of the three lignin concentrations, media
supplemented with 0.25 % (w/v) exhibited the most efficient degradation of the dye. It
223
is very likely, that higher concentrations of lignin added to media could have an inhibitory
effect on fungal growth. Matjuškova et al., (2017) in a recent study on the effect of lignin-
containing media on fungal growth observed, that inclusion of 0.25 %-0.5 % (w/v) of
Kraft lignin resulted in increased growth while higher concentrations (1 % and 2.5 %
(w/v)), led to strong suppression of mycelium growth.
The ligninolytic capability of T. harzianum was investigated further in liquid media at pH

5 and pH 7, to test the effect of the different pH on fungal growth in a liquid environment.
As the growth in dark brown lignin supplemented broth could not be determined (Tables
4.8 and 4.9), liquid culture was assayed for ligninolytic activity test. Decolourisation of
methylene blue was observed in all spent culture supernatants at pH 5 with highest
activity observed with lignin supplemented at 0.5 % (w/v) and the lowest in broth
supplemented with 2 % (w/v) (Table 4.10) (Appendix, Figures 6 and 7). These results could
potentially support the theory of the inhibitory effect of lignin at high concentrations.
The lack of methylene blue decolourisation at pH 7, was either because there was no
ligninolytic enzymes produced in culture at this pH or the enzymes were not active at
this pH.
Bacteria are also worthy of being studied for their ligninolytic potential due to their
immense environmental adaptability and biochemical versatility (Bholay et al., 2012).
Based on positive results, suggesting ligninolytic capability of T. harzianum in the
presence of CIMV lignin, the same screening assays were carried out for bacterial strains.
Two sets of experiments were performed. The first involved screening P. putida, P.
saccharophila and P. fluorescens for the ability to grow on lignin containing solid minimal
media and their ability to decolourise dye as an indicator for the expression of ligninolytic
enzymes. The aim of the second set was to determine the potential growth and presence
of ligninolytic enzymes in liquid media.
The results of screening carried out on M9 solid media supplemented with 0.25 % (w/v)
of CIMV lignin showed, that none of the strains were able to utilize lignin as a carbon and
energy source for growth (Table 4.11) (Appendix, Figure 8). In order to investigate the
presence/lack of ligninolytic capability, all strains were inoculated onto M9 agar media
supplemented with either methylene blue alone, methylene blue and glucose, or
224
methylene blue and CIMV lignin as the sole carbon and energy source, and screened for
growth and formation of clear zones. None of the screening media revealed expression
of ligninolytic enzymes (Table 4.12) (Appendix, Figures 9, 10 and 11). A possible
explanation is that the dye-decolourizing enzyme activity of Pelomonas saccharophila
and Pseudomonas species on solid environment could be limiting, especially when grown
on minimal media. Data presented by Tian et al., (2016) showed, that degradation of
methylene blue by gram negative bacteria, including Pseudomonas sp., occurred in both
in minimal and rich solid media. However, the amount of dye added to the media was
approximately two to three times higher (0.10-0.15 g/L) than in experiments described Commented [AS[116]: we clarified this
Commented [AW[117]: not sure why higher dye content
here. Similar results were also observed by Sasikumar et al., (2014). In their study, should result in more decolorisation
ligninolytic activity of Pseudomonas sp. was observed in rich media supplemented with
a high concentration of methylene blue (0.25 g/L). In another study, Bandounas et al.,
(2011) showed, that Pseudomonas sp. decolourized methylene blue added to minimal
media at low concentrations (0.025 g/L) but in the presence of 2 additional carbon and
energy sources. Therefore, it is very likely, that a combination of an appropriate amount
of carbon and energy source in the form of dye or other media components are
necessary for ligninolytic enzyme production on solid media. It’s also possible, that
decolourisation would have been observed, if plate were incubated for the longer
period. It is of interest, that while P. putida and P. fluorescens showed clear growth on
plates with M9 minimal media, methylene blue and glucose within 3 days of incubation,
no decolourisation was observed. This is most likely because methylene blue was not
used as a carbon and energy source and the growth was related to the presence of
glucose. The lack of P. saccharophila growth indicated, that cells had died before they
were transferred to the plate (Appendix, Figure11).
Since no bacterial growth was observed on solid media in the presence of CIMV lignin,
further experiments were carried out to investigate growth in liquid media. The results
of the analysis revealed, that P. putida, P. saccharophila and P. fluorescens were able to
utilize CIMV as a carbon and energy source for growth within 96 hours incubation (Figure
4.2).
These studies have determined conditions for demonstrating utilisation of lignin and
expression of ligninolytic enzymes. Although solid state fermentation is particularly
225
useful for growing filamentous fungi and many species do not sporulate efficiently in
liquid culture (Bailey et al., 2009) it is proposed, that fungal and bacterial growth in liquid
culture would be more practical in the context of further analysis of lignin break-down
products or enzyme purification. Preliminary experiments revealed, that 0.25 % (w/v) of
lignin is an optimal level for supplementing media as higher amounts could lead to
suppression of fungal and bacterial growth.
Screening tests carried out during this study showed, that T. harzianum, P. putida, P.
saccharophila and P. fluorescens have the ability to utilize CIMV lignin as the sole carbon
and energy source for growth both in solid and liquid media. It must be noted, that
results presented in Chapter 3 (sections 3.4.1.1 and 3.5.1.1) showed that trace amounts
of hemicellulose are still present in CIMV lignin, however levels were very low and cannot
account for observed growth. Overall results from this study suggested, that CIMV lignin
could be a basis tool for screening microorganisms which may have application in
industrial biotechnology. Microbial lignin degradation could be harnessed for lignin
bioconversion to aromatic chemicals with potential applications including renewable
plastic production (Mycroft et al., 2015). However, it is important to consider that
technical lignin represents much a more complex and challenging substrate than model
compounds used in lignin degradation studies. Hence, understanding the metabolism of
these more complex mixtures by microbial strains would be a logical subsequent
necessary step for real valorisation of lignin (Ravi et al., 2017).
226
4.6 CONCLUSIONS
Filamentous fungi such as T. harzianum and the gram negative bacteria: P. putida, P.
saccharophila and P. fluorescens were screened for the ability to grow in minimal media
supplemented with different amounts of Miscanthus giganteus biomass and CIMV
organosolv lignin as the sole carbon and energy source. The results of growth
experiments revealed, that T. harzianum was able to utilize lignocellulosic material as a
carbon and energy source in liquid media. The same strain was also capable of utilizing
organosolv lignin on solid and liquid media. Although, the growth of T. harzianum on
organosolv lignin in liquid media could not be determined, there was an evidence of
ligninolytic activity in the spent culture media. Studies with bacteria showed, that none
of the species was able to grow on minimal solid media supplemented with organosolv
lignin, however the same strains were capable of utilising of organosolv lignin as C-
carbon in liquid media. This indicated, that investigated bacteria cannot utilise lignin
substrate as a carbon and energy source on solid minimal media. The overall assessment
demonstrated, that T. harzianum, P. sacharophila. P. putida and P. fluorescens strains
are capable of metabolising organosolv lignin. This makes them good candidates for
applications in the production of lignin-based products.
227
Chapter 5
228
Chapter 5: MICROBIAL DEGRADATION OF LIGNIN: INSIGHTS INTO
MECHANISMS OF FUNGAL-AND BACTERIAL-MEDIATED
ORGANOSOLV LIGNIN BREAK-DOWN
5.1 ABSTRACT
A detailed study of ligninolytic capability of T. harzianum, P. putida, P.saccharophila and Commented [AW[118]: In full
P. fluorescens was carried out by monitoring growth on CIMV organosolv lignin and
characterisation of lignin degradation products. The main aim of this study was to
provide insights into the mechanism of microbial-mediated organosolv lignin
degradation, in order to understand the biochemistry and structure-function
relationships of different pathways involved in this process. Bacterial and fungal growth
was monitored over 360 hours in M9 liquid media supplemented with 0.25 % (w/v) of
CIMV lignin. Qualitative and quantitative analysis of samples taken at different time
intervals during growth was carried out by LC-PDA-ESI/MSn. Lignin degradation patterns
were compared between the species and identified compounds were analysed in the
context of a potential source of high-value products. Analysis of cell-free culture broth
extracts revealed the presence of ferulic acid, coumaric acid, 4-O-5’-coupled diferulic
acid, 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (GHP), 3-hydroxy-1(4-
hydroxy-3,5-dimethoxyphenyl)-1-propanone (SHP), S(8-5)FA and G(8-5)FA. The
identified compounds indicated cleavage of the ester and β-O-4 ether linkages and
catabolism of phenylcoumarans. Characterisation of the lignin degradation patterns
showed, that the ligninolytic capability of T. harzianum differed from bacteria. The
fungus investigated in this study produced very low amount of hydroxycinnamic acids
and other break-down products were only observed with the inclusion of an extra carbon
and energy source .Of the four strains tested, P. saccharophila was proposed to be the
most promising organism for bioconversion of lignin. The high amount of ferulic acid and
GHP produced by this bacteria indicated potential for production of high-value chemicals
with application in the food and chemical industry and building blocks for the production Commented [AW[119]: Check – what food applications?
of polyesters, polycarbonates, epoxy and urethane resins.
Keywords: CIMV lignin degradation • Lignonolytic bacteria • Liginolytoc fungi • Ferulic

acid • 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (GHP)
229
5.2 INTRODUCTION
Lignin is the most abundant aromatic renewable resource on earth. To enable the
application of lignin as a raw material, it is necessary to degrade or modify these
molecules by physical, chemical, and/or biological methods (Numata and Morisaki,
2015).
Recently, bioconversion of lignin with microbes emerged as an alternative lignin

valorisation approach with significant potential (Xie et al., 2016). Since lignin is composed
of phenylpropanoid units, the oxidative degradation of lignin could potentially release
low molecular weight compounds (Bugg and Rahmanpour, 2015). Such lignin
degradation products would be a valuable source of aromatics for chemical, food, and
pharmaceutical industries (Helmich et al., 2016).
Biodegradation of lignin using ligninolytic enzymes has been extensively studied in the
last couple of years. A considerable amount of literature has highlighted, that ligninolytic
enzymes have better properties compared to other depolymerisation methods in terms
of improving the overall efficiency for production of chemicals (Hofrichter, 2002., Wong,
2009; Lange et al., 2013; Asina et al., 2017). This is because, they can react selectively at
certain linkage positions on the lignin structure and facilitate lignin depolymerisation
under very mild conditions (Ohta et al., 2017; Asina et al., 2017). These reactions can
also result in the creation of new functional groups.
In order to take the advantage of bioconversion of lignin, especially to derive streams

aromatic compounds for use in value-added products, further insight into lignin
metabolism is needed (Fisher and Fong, 2014). Currently, there are ongoing studies by Commented [AW[120]: Make sure wording is different to
source
Tim Bugg’s group (University of Warwick, UK) to investigate the products produced by
bacterial and fungal degradation of lignin. Compounds identified in early studies
suggested a range of enzymes attacking different lignin substructures (Bugg et al., 2011;
Taylor et al., 2012; Bugg and Rahmanpour, 2015). However, knowledge of pathways
utilized for catabolism of lignin-derived components is still incomplete (Fisher and Fong,
2014). According to Asina et al., 2017, fungi and bacteria use fundamentally different
strategies in lignin utilization. Hence, there is a need to look at variation in product
230
formation with different microorganisms to determine which is best suited for specific
industrial applications (Ahmad, 2010).
Reports on microbial degradation of sulphur-free lignin such as organosolv are currently

limited to only a few studies. Taylor, (2013) investigated the ligninolytic activity of
Sphingobacterium sp and M. phyllosphaerae. In turn, Rodriguez et al., (1994) and Amaral
et al., (2012), reported on the capability to degrade organosolv lignin by Penicillum
chrysogenum and Aspergillus niger, respectively. However, research on organosolv
lignin degradation products and possible pathways involved in this process have not
been published to date. As described in Chapter 4, T. harzianum, P. putida, P.
saccharophila, and P. fluorescens could utilise CIMV organosolv lignin as a carbon and
energy source and provided evidence of ligninolytic enzyme production visualised by dye
decolourisation assays. Hence, it was postulated that an analysis of break-down products
produced by the different strains could help to gain a further understanding of the
metabolism of lignin and develop a process for the valorisation of organosolv lignin. The
main aim of the study presented in this Chapter was to provide insights into the
mechanism of microbial-mediated organosolv lignin degradation. The ligninolytic
capability of Trichoderma harzianum, Pseudomonas putida, Pelomonas saccharophila
and Pseudomonas fluorescens was examined by carrying out growth experiments in M9
and YNB liquid media supplemented with 0.25 % (w/v) of lignin. Further work focused on
characterization of lignin degradation products. The quantitative analysis of each
compound recovered at different time intervals during 360 hours incubation was carried
out on LC-PDA-ESI/MSn. Lignin degradation patterns were compared between the
species to understand the biochemistry and structure-function relationships of different
pathways involved in bioconversion process. The identified compounds were discussed
in the context of potential source of high-value chemicals or building blocks for the
production of bio-based materials.
231
described below. The procedures used for growth experiment were carried out under
aseptic conditions, using sterile chemicals and laboratory equipment. Unless otherwise
stated, all plastic ware and commonly used chemicals of the highest grade available were
purchased from Fisher Scientific (Lecicestershire, UK) or Sigma-Aldrich Company Ltd
(Dorset, UK). Any glassware used in the carried out procedures were of Duran or Pyrex
brand. For all steps, purified water, (Elga Purelab Classic UV, VWS UK Ltd, 18.2 mega
ohm-cm resistivity at 25 °C) was used unless otherwise stated.
5.3.1 FEEDSTOCKS
The feedstock used in this study was Compagnie Industrielle de la Matière végétale
(CIMV) organosolv wheat straw lignin as a standard, which was kindly supplied by CIMV Commented [AW[121]: In full first time in methods-
Company, Pomacle, Champagnes-Ardennes, France.
The fungal and bacterial species used in this study were: Trichoderma harzianum,
Pseudomonas putida, Pelomonas saccharophila and Pseudomonas fluorescens. T.
harzianum (coded TH), cultured on YPD agar media was kindly provided by Wageningen
UR, Food & Biobased Research, Wageningen, The Netherlands. P. putida (NCIMB 8248;
coded PP), P. saccharophila (NCIMB 8570; coded PS) and P. fluorescens (NCIMB 9397;
coded PF) were Aberystwyth University (AU) in-house strains maintained in glycerol
stocks by IBERS, AU, Wales, UK.
5.3.3 CHARACTERISATION OF LIGNIN METABOLISING MICROORGANISMS

5.3.3.1 Growth of T. harzianum, P. putida, P. saccharophila, P. fluorescens in YNB and M9
liquid media supplemented with CIMV lignin
All cultures were grown in duplicate, in 250-mL shake flasks, in 100 mL of YNB minimal
media (prepared as described in section 4.3.5.1) (pH 5, optimal for fungal growth) and
100 mL of M9 minimal media (prepared as described in section 4.3.5.2) (pH 7, optimal for
bacterial growth), supplemented with CIMV lignin as the sole carbon and energy source.
232
In order to avoid complexing of lignin with other media components during autoclaving, Commented [AW[122]: I think dark colour formed when
added directly to media is due to reactions with other components
CIMV lignin was freeze-dried, autoclaved and added separately to sterile media in the in medium
following amount: 0.25 % (w/v). Single colonies from original LB agar plates (prepared as
described in section 4.3.3) were inoculated in 10 mL of fresh LB liquid media, incubated
at 30 oC with 180 rpm and grown to OD590 values of: 1.1 (P. putida), 0.75 (P.
saccharophila) and 0.8 (P. fluorescens). YNB liquid media supplemented with lignin, was
inoculated with agar blocks of 5mm x 5mm taken from a mycelial mat (prepared as
described in section 4.3.3) and M9 liquid media supplemented with lignin was inoculated
with 2 mL of inoculum of bacterial strains. Cultures were mixed thoroughly, by keeping
the flasks on a rotary shaker at 180 r. p. m at 30 oC for 360 hours. Aliquots of each culture
(1mL) were removed after 4, 8, 18, 24, 48, 72, 96, 144, 216, 288, 360 hours and the OD 590
was monitored using CO8000 cell density meter (Biochrom, Wpa, Cambridge, UK). All
readings were taken in triplicate and the cultures exhibiting readings above 1.0 OD590
were diluted 2-5-fold in the appropriate culture media. Two control treatments were
also included, a positive control with microorganisms grown in media supplemented with
0.25 % (w/v) of lignin and 0.25 % (w/v) of glucose and a negative control with media
without carbon and energy source.
5.3.4 ANALYSIS OF LIGNIN BREAK-DOWN PRODUCTS AND LIGNIN-RELATED PHENOLICS BY LIQUID-
CHROMATOGRAPHY- PHOTODIODE ARRAY DETECTION- TANDEM MASS SPECTROMETRY
5.3.4.1 Incubation time course

Duplicate 2 mL samples were removed from the fermentation culture after 4, 8 ,18, 24,
48, 72, 96, 144, 216, 288, 360 hours and transferred into 2mL centrifuge tubes. The
samples were centrifuged at 14.000 x g for 10 minutes and the supernatants were stored
at -80 oC for further analysis.
5.3.4.2 Samples preparation and analysis

Samples preparation and analysis was carried out adapting the method described by
Hauck et al., (2014). Prior to analysis, samples were partially purified by Solid Phase
Extraction (SPE) using Sep-Pak C18 catridges (500 mg; 3cc) (Waters Ltd, Elstree, UK). The
cartridges were activated with 4 mL of 100 % methanol, followed by 4 mL of 5 % acetic
acid. Samples were then loaded, washed with 2.5mL of water, eluted with 4 mL of 100
% methanol, dried down at 60 oC using rotary evaporation and dissolved in 0.5 mL of 70
233
% methanol. Samples were analysed by reverse-phase liquid chromatography with
online photodiode array detection and electrospray ionisation-ion trap mass
spectrometry (LC-PDA-ESI/MSn). Analysis was carried out on a Thermo Finnigan LC-MS
system (Thermo Electron Corp., Waltham, MA, USA) comprising a Finnigan PDA plus
detector, a Finnigan LTQ linear ion trap with ESI source and the column used was a 3.9
mm x 100 mm i.d., 4 µm, C18 Nova-Pak (Waters) with temperature maintained at 30 oC.
The PDA scan range was set to 240-400 nm and the sample injection volume was typically
10 µL and the flow rate was 1mL min-1 with 100 µL min-1 going to the mass spectrometer.
The mobile phase consisted of water with 0.1 % formic acid (solvent A) and methanol
with 0.1 % formic acid (solvent B). The column was equilibrated with 95 % solvent A and
the percentage of B increased linearly to 65 % over 60 minutes. Mass spectra parameters
were as follows: sheath gas 30 arbitrary units, auxiliary gas 15 arbitrary units and sweep
gas 0 units, spray voltage -4.0 kV in negative and 4.8 kV in positive ionisation, capillary
temperature 320 oC, capillary voltage -1.0 V and 45 V respectively, tube lens voltage -68
and 100 V respectively, and normalized collision energy (CE) 35 %. All samples were run
in duplicate technical replicates. The data were recorded using the Xcalibur 2.0.0
software package (Fisher Scientific, Leicestershire, UK). Products were identified based
on standards or information found in the literature. Compounds quantification was
determined by comparison with high-grade standards: (a) ferulic acid for ferulic acid, 3-
hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone, G (8-5)FA and 4-0-5’-coupled
diferulic acid, b) coumaric acid for coumaric acid, c) sinapic acid for S(8-5)FA, assessing
the area under the compound peak in the UV chromatogram. Standard curves (Figures
5.1, 5.2 and 5.3) were generated using a series of injection volumes of appropriate
standards.
234
Ferulic acid
700000
600000
500000
Peak area
400000 y = 1E+06x + 87521

300000 R² = 0.996
ferulic acid
200000
100000
0
0 0.1 0.2 0.3 0.4 0.5 0.6
Ferulic acid concentration (ug/injection)
Figure 4.31: Standard curve for ferulic acid.
Coumaric acid
7000000
6000000
5000000
Peak area
4000000 y = 1E+07x + 695298

3000000 R² = 0.9962
coumaric acid
2000000
1000000
0
0 0.1 0.2 0.3 0.4 0.5 0.6
Coumaric acid concentration ug/injection
Figure 4.42: Standard curve for coumaric acid.
235
Sinapic acid
1400000
1200000
1000000
Peak area
800000 y = 2E+06x + 109307

600000 R² = 0.9955
sinapic acid
400000
200000
0
0 0.1 0.2 0.3 0.4 0.5 0.6
Sinapic acid concentration ug/injection
Figure 4.53: Standard curve for sinapic acid.
236
5.4 RESULTS
5.4.1 FUNGAL AND BACTERIAL GROWTH PATTERNS WITH ORGANOSOLV LIGNIN AS A CARBON AND ENERGY
SOURCE
5.4.1.1 Growth of T. harzianum, P. putida, P. saccharophila, P. fluorescens in YNB and M9 Commented [AW[123]: Screened in previous chapter
liquid media supplemented with CIMV lignin

T. harzianum, P. putida, P. saccharophila and P. fluorescens were inoculated into YNB
and M9 liquid media supplemented with CIMV lignin as the sole carbon and energy
source at pH 5 (fungi) or pH 7 (bacteria) and 30 oC. Based on results from Chapter 4, a
lignin concentration of 0.25 % (w/v) was added to the media (as described in section
5.3.3.1). Fungal growth was evaluated visually at 0, 4, 8, 18, 24, 48, 72, 96, 144 hours and
then every 72 hours over a time course of 360 hours. Cell growth was also monitored at
OD590 at the same time intervals. Positive control samples were included with addition
of glucose (0.25 % w/v) to YNB/M9 media supplemented with lignin. The results showed,
that T. harzianum was capable of utilizing lignin as the sole carbon and energy source for
its growth confirmimg observations described in Chapter 4. Although, there was no
growth observed in the first 48 hours of incubation, a low level of growth was observed
between 48-360 hours. In turn, moderate growth was detected between 144 and 360
hours in media extra supplemented with 0.25 % (w/v) of glucose (Table 5.1). All bacterial
strains exhibited good growth in M9 liquid media supplemented with CIMV lignin (0.25
% w/v) and glucose (0.25 % w/v), with the highest growth rate (log phase) between 0-18
hours.Whilst all strains were capable of good growth in M9 media with lignin and
glucose, some strains grew more rapidly. P. saccharophila and P. putida showed similar
growth pattern, reaching the highest OD590, 2.13 and 2.15, respectively, at 18 and 24
hours. In contrast, P. fluorescens grew less rapidly, reaching the highest OD590, 1.75, at
24 hours. P. putida and P. fluorescens cells showed a decrease in OD590 of 0.15 and 0.30
respectively between 24-48 hours, while P. saccharophila showed a decrease in OD590 of
0.13 between 18-24 hours. In the case of P. putida a gradual decrease in OD590 was
observed up to 144 hours. After that, P. putida cells exhibited stationary phase up to the
end of 360 hours period. In turn, a decrease in OD590 of P. saccharophila cells was
observed up to 288 hours. This strain showed an increase in OD 590 of 0.35 in the last Commented [AS[124]: Can’t confirm the cell death unless you
test viability
72 hours of fermentation, suggesting switch to a different metabolic pathway. P. In case of P putida graduate decrease was observed up to 144, in
case f p. Saccharophila upo to 288 dont say anything about p.
fluorescens remained in stationary phase between 48-360 hours. (Figure 5.4 A). Analyses fluorescens
237
of optical density for bacterial strains grown in the presence of CIMV lignin as the sole
carbon and energy source showed much lower levels of growth than observed with
media supplemented with both CIMV lignin and extra glucose. The growth patterns also
showed variation with strains. P. putida and P. fluorescens showed good growth in the
first 18 hours of incubation, followed by a short stationary phase between 18-48 hours.
After that, bacteria showed a steady slow rate of growth up to 360 hours. P.
saccharophila showed the highest rate of growth in the first 18 hours and in common
with the other two strains, a short stationary phase (between 18-48 hours). Some growth
was observed between 48-72 hours with a stationary phase up to 144 hours of
incubation after which point bacteria continued to grow at a slow rate. P. putida, P.
saccharophila and P. fluorescens cultures grew to the same extent over the course of
fermentation with respective OD590 values of 0.77, 0.77 and 0.75 observed at 360 hours
(Figure 5.4 B). The maximum OD590 observed with cultures of Pelomonas strain and
Pseudomonas sp. in M9 media in the presence of CIMV lignin was two to three fold lower
than the level observed in the presence of CIMV lignin and glucose (Figures 5.4 A and B).
However, all tested strains were able to grow in the presence of CIMV lignin as the sole
carbon and energy source, indicating their capability to produce enzymes necessary for
degradation of lignin polymer. The culture broth was further investigated for lignin
break-down products and any other lignin-related phenolics.
238
Table 4.131: Growth of T. harzianum in YNB liquid media supplemented with 0.25 % (w/v) of CIMV lignin
at 30 oC . Control -: YNB liquid media and inoculum, Control +: YNB liquid media with 0.25 % (w/v) of
CIMV lignin, 0.25 % (w/v) of glucose and inoculum.
Treatment Extent of growth
Time
(hours)
0 4 8 18 24 48 72 96 144 216 288 360
0.25 % (w/v) lignin

- - - - - + + + + + + +
C+ - - - - - + + + ++ ++ ++ ++
C- - - - - - - - - - - - -
, ++ = moderate growth, + = poor growth, - = no growth,
239
A
Commented [AW[125]: remove lig + glu

2.40
2.20 tae out the line , make the letters in bold , change to 590
2.00
Optical density @ 590nM
1.80
1.60
1.40
PP
1.20
1.00 PS
0.80 PF
0.60
0.40
0.20
0.00
0 40 80 120 160 200 240 280 320 360 400
Time (hours)
1.00
Optical density @ 590nM
0.80
0.60
PP
0.40 PS
PF
0.20
0.00
0 40 80 120 160 200 240 280 320 360 400
Time (hours)
Figure 4.64: Growth of P. putida, P. saccharophila and P. fluorescens in M9 liquid media supplemented
with: A) 0.25 % (w/v) of CIMV lignin, B) 0.25 % (w/v) of CIMV lignin, 0.25 % (w/v) glucose for 360 hours at
30 oC and pH 7. Error bars represent standard deviation (SD±) of the mean (n=3).
240
5.4.2 ANALYSIS OF LIGNIN BREAK-DOWN PRODUCTS AND LIGNIN-RELATED PHENOLICS
5.4.2.1 Product identification
Fermentation broth from different time intervals: 4, 8, 18, 24, 48, 72, 96, 144, 216, 288,
360 hours was purified (as described in section 5.3.4) and analysed by LC-PDA-ESI/MSn.
Analysis of cell-free broth extracts revealed the presence of low molecular weight
phenolic compounds including the following lignin-related molecules: coumaric acid,
ferulic acid, 4-0-5’-coupled diferulic acid and lignin break-down products: S(8-5)FA, G (8-
5)FA),3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone,3-hydroxy-1-(4-hydroxy-
3,5dimethoxyphenyl)-1-propanone. Figure 5.5 represents a typical UV chromatogram
showing phenolic profile of culture broth extract. Compounds for which no standards
are available, were tentatively identified based on fragmentation patterns and
information from the literature. Molecuar structures and fragmentation ions are
presented in Table 5.2.
241
Figure 4.75: Example of HPLC phenolic profile of P. saccharophila culture broth extract supplemented with 0.25 % (w/v) of CIMV lignin after 48 hours of incubation. Detection
at 340 nm. Peaks: see Table 5.2.
242
Table 4.142: Lignin break-down products and lignin-related phenolics identified in cell-free extracts after incubation of P. putida, P. saccharophila, P. fluorescens and T.
harzianum in M9 liquid media supplemented with 0.25 % (w/v) of lignin for 360 hours at 30 oC.
RT UV max [M-H]- -MS2[M-H]- Compound Proposed structure Reference

(nm) (m/z) (m/z)
Reiter et al.,
3-hydroxy- (2013)
7.8 275 195 180 1-(4-
hydroxy-3-
methoxyph
enyl)-1-
propanone
(1)
Reiter et al.,
3-hydroxy- (2013)
8.6 295 225 210 1-(4-
hydroxy-
3,5-
dimethoxyp
henyl)-1-
propanone
(2)
Carvalho et al.,
(2015)
9.2 309 163 119 Coumaric
acid
(3)
243
Carvalho et al.,
(2015)
10.2 323 193 178 Ferulic acid
(4)
Morreel et al., Field Code Changed

(2010)
14.0 325 401 383,371 S(8-5)FA
(5)
Morreel et al.,
(2010)
14.2 320 371 353,341, G (8-5)FA
327 (6)
(Bunzel et al.,
14.9 283, 385 341 4-0-5’- 2000)
323 coupled
diferulic
acid
(7)
244
5.4.2.2 Analysis of lignin-related phenolics by HPLC-PDA-ESI-MSn
Coumaric acid
Compound 3 produced an ion at m/z 163 in MS1 in negative mode, which corresponds
to coumaric acid, and was observed in all culture broth samples. However, the results of
the analysis showed, that accumulation of this product varied with microorganism and
carbon source over a time course of 360 hours (Figure 5.6). The most rapid rate of Commented [AW[126]: Refer to figure
accumulation in culture broth with lignin as the sole carbon and energy source for P.
saccharophila, P. fluorescens and T. harzianum was observed between 24-48 hours
(Figure 5.7 B, C and D). In contrast, P. putida cultures showed greatly reduced
accumulation of coumaric acid indicating different metabolic pattern. An initial decrease
in coumaric acid content was observed in the first 8 hours and a low level of
accumulation was noticed between 48-96 hours of incubation (Figure 5.7 A). The highest
concentration was detected in P. saccharophila lignin supplemented broth, at 48 hours
(0.017 mg/mL). In turn, the lowest level of accumulation of coumaric acid was observed
in the case of P. putida grown on lignin as the only carbon and energy source (maximum
concentration at 96 hours: 0.0009 mg/mL). All bacterial strains had metabolized
coumaric acid within 216 hours while in comparison, accumulated levels persisted in T.
harzianum cultures remaining relatively stable up to 360 hours (Figure 5.6 A). All species
exhibited very similar patterns of coumaric acid accumulation with and without the
inclusion of glucose (Figure 5.6 B/Figure 5.7 A, B, C and D). Although, adding glucose
generally decreased coumaric acid content in the case of P. putida and P. saccharophila
lignin supplemented broth (notably between 72-96 hours and 48-96 hours, respectively)
the compound had been fully metabolised within 216 hours (Figure 5.7 A and B). Commented [AW[127]: I wouldn’t go into too much detail
here as these differences may not be significant
245
0.016 A 0.016 B
Compound (mg/mL)
Compound (mg/mL)
0.012 0.012
PP PP
0.008 PS 0.008
PS
PF PF
0.004 0.004
TH TH
0 0
0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
Time (hours) Time (hours)
Figure 4.86: Coumaric acid content (mg/mL) in culture broths of P. putida, P. saccharophila, P. fluorescens and T. harzianum strains supplemented with CIMV lignin (0.25 %
w/v) alone (A) and in the presence of glucose (0.25 % w/v) (B). Error bars represent standard error (SE±) of the mean (n=2).
246
0.002
A 0.02 B
0.0016
Compound (mg/mL)
Compound (mg/mL)
0.016
0.0012
0.012
PP+lig PS+lig
0.0008
PP+lig+gluc 0.008 PS+lig+gluc
0.0004
0.004
0 0
0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
C 0.016 D
0.016
Compound (mg/mL)
Compound (mg/mL)
0.012
0.012
PF+lig 0.008 TH+lig
0.008
PF+lig+gluc TH+lig+gluc
0.004 0.004
0 0
0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
Figure 4.97: Coumaric acid content (mg/mL) in culture broths of A) P. putida, B) P. saccharophila, C) P. fluorescens, D) T. harzianum strains supplemented with CIMV lignin
alone and in the presence of glucose over a time course of 360 hours. Error bars represent standard error (SE±) of the mean (n=2).
247
Ferulic acid
Compound 4 produced an ion at m/z 193 in MS1 in negative mode, which corresponds to
ferulic acid, and was observed in all culture broth samples. However, as with coumaric
acid, the accumulation of this product varied with fungal and bacterial species, and with
carbon source, over a time course of 360 hours (Figure 5.8). The most rapid rate of
accumulation in culture broth with lignin as the sole carbon and energy source for P.
putida, P. saccharophila, P. fluorescens and T. harzianum was observed between 48-96
hours, 24-48 hours, 24-72 hours and 24-48 hours respectively (Figure 5.9 A, B, C and D). Commented [AW[128]: Refer to figure when you give data
The highest concentration was detected in P. putida and P. saccharophila cultures, with
lignin as the sole carbon and energy source, at 144 hours (0.046 mg/mL). In turn, the
lowest level of accumulation of ferulic acid was observed in the case of T. harzianum
grown on lignin as the sole carbon and energy source (maximum concentration at 360
hours: 0.018 mg/mL). Ferulic acid content decreased notably in P. putida, P.
saccharophila and P. fluorescens lignin supplemented broths, by 216 hours. Thereafter,
total ferulic acid content in bacterial broths remained stable up to 360 hours. Levels were
also relatively constant after 144 hours in T. harzianum culture broth (Figure 5.8 A).
Bacterial strains exhibited different patterns of ferulic acid accumulation in the presence Commented [AW[129]: Better to compare all bacterial strains
of glucose as an extra carbon and energy source (Figure 5.8 B). Inclusion of glucose
markedly decreased ferulic acid content in the case of P. putida grown on lignin as the
only carbon and energy source between 24-216 hours. The lower content over a time
course of 360 hours was also observed with P. saccharophila lignin supplemented broth.
Ferulic acid notably decreased after 96 hours. Thereafter, a slower rate of increase was
observed in the latter stage of fermentation. In the case of P. fluorescens culture
supplemented with lignin and glucose as a carbon and energy source, the compound was
completely metabolised by 360 hours (Figure 5.9 A, B, C). No evident differences in ferulic
acid accumulation were observed with T. harzianum in the presence of glucose as an
extra carbon and energy source (Figure 5.9 D).
248
0.06 0.06
A B
Compound (mg/mL)
Compound (mg/mL)
0.04 0.04
PP PP
PS PS
0.02 PF 0.02
PF
TH TH
0 0
0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
Figure 4.108: Ferulic acid content (mg/mL) in culture broths of P. putida, P. saccharophila, P. fluorescens and T. harzianum strains supplemented with CIMV lignin (0.25 % w/v)
alone (A) and in the presence of glucose (0.25 % w/v) (B). Error bars represent standard error (SE±) of the mean (n=2).
249
0.06 0.06
A B
Compound (mg/mL)
Compound (mg/mL)
0.04 0.04
PP+lig PS+lig
0.02 0.02
PP+lig+gluc PS+lig+gluc
0 0
0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
0.06 0.06
C D
Compound (mg/mL)
Compound (mg/mL)
0.04 0.04
0.02 PF+lig TH+lig

0.02
PF+lig+gluc TH+lig+gluc
0
0 50 100 150 200 250 300 350 400 0
Time (hours) 0 50 100 150 200 250 300 350 400
Time (hours)
Figure 4.119: Ferulic acid content (mg/mL) in culture broths of A) P. putida, B) P. saccharophila, C) P. fluorescens, D) T. harzianum strains supplemented with CIMV lignin alone
an in the presence of glucose over a time course of 360 hours. Error bars represent standard error (SE±) of the mean (n=2).
250
4-0-5’-coupled diferulic acid
Compound 7 produced an ion at m/z 385 in MS1 in negative mode, which corresponds to
4-0-5’-coupled diferulic acid, and was observed in all culture broth samples. However,
the results of the analysis showed, that accumulation of this product varied again with
microorganism and carbon and energy source over a time course of 360 hours (Figure Commented [AW[130]: Refer to figure
5.10). The most rapid rate of accumulation in culture broth with lignin as the sole carbon
and energy source for P. putida, P. saccharophila and P. fluorescens was observed
between 48-96 hours, 18-48 hours and 24-48 hours, respectively (Figure 5.11 A, B and
C). In turn, T. harzianum grown on lignin as the sole carbon and energy source, showed Commented [AW[131]: Keep to more general trends
very low levels of accumulation with the highest rate observed between 24-48 hours of
incubation (Figure 5.11 D). The highest concentration was detected in P. saccharophila
lignin supplemented broth at 144 hours (0.024 mg/mL). In turn, the lowest level of
accumulation of 4-0-5’-coupled diferulic acid was observed in the case of T. harzianum
grown on lignin as the sole carbon and energy source (maximum concentration at 360
hours: 0.006 mg/mL). 4-0-5’-coupled diferulic acid content decreased markedly in P.
putida and P. saccharophila cultures after 144 hours, followed by a slight increase at the
latter stage of incubation. In the case of P. fluorescens lignin supplemented broth, the
levels remained relatively stable between 96-288 hours, with a slight increase observed
in the final 72 hours of the incubation. In the case of T. harzianum growth on lignin as
the sole carbon and energy source, the content of 4-0-5’-coupled diferulic acid remained
low throughout the incubation period with trends difficult to establish as levels were
below accurate detection limit. (Figure 5.10 A). P. putida and P. fluorescens exhibited very
similar patterns of 4-0-5’-coupled diferulic acid accumulation in the presence of glucose
as an extra carbon and energy source (Figure 5.10 B). Although inclusion of glucose
generally resulted in a lower content of 4-0-5’-coupled diferulic acid notably between 72
and 216 hours, levels were observed to increase in the final 144 hours of the incubation
(Figure 5.11 A and C). Compared to other species, P saccharopila and T. harzianum
exhibited slightly different pattern of 4-0-5’-coupled diferulic acid accumulation. In the
case of P. saccharophila culture supplemented with lignin and glucose as a carbon and
energy source, the initial increase of 4-0-5’-coupled diferulic acid content in the first 72
hours was followed by a more marked reduction by 96 hours. In general, inclusion of Commented [AW[132]: I think you can’t read to much into
this- looks like noise and t harzianum is too low to be accurate
251
glucose decreased product accumulation between 96-216 hours (Figure 5.11 B). In turn,
in the case of T. harzianum culture supplemented with lignin as the sole carbon and
energy source, adding glucose markedly increased 4-0-5’-coupled diferulic acid content
in the first 72 hours. However, levels were again very low and on the borderline of
detection limit (Figure 5.11 D).
252
0.024
A B
Compound (mg/mL)
Compound (mg/mL)
0.024
0.016
0.016 PP PP
PS PS
0.008
0.008 PF PF
TH TH
0 0
0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
Figure 4.1210: 4-0-5’-coupled diferulic acid content (mg/mL) in culture broths of P. putida, P. saccharophila, P. fluorescens and T. harzianum strains supplemented with CIMV
lignin (0.25 % w/v) alone (A) and in the presence of glucose (0.25 % w/v) (B). Error bars represent standard error (SE±) of the mean (n=2).
253
0.03 0.03
A B
Compound (mg/mL)
Compound (mg/mL)
0.02 0.02
PP+lig PS+lig
0.01
0
0 50 100 150 200 250 300 350 400 0
Time (hours) 0 50 100 150 200 250 300 350 400
Time (hours)
0.03 0.007
Compound (mg/mL)
Compound (mg/mL)
C 0.006 D
0.02 0.005
0.004
PF+lig 0.003 TH+lig
0.01 0.002
PF+lig+gluc TH+gluc+lig
0.001
0 0
0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
Figure 4.1311: 4-0-5’-coupled diferulic acid content (mg/mL) in culture broths of A) P. putida, B) P. saccharophila, C) P. fluorescens, D) T. harzianum strains supplemented with
CIMV lignin alone and in the presence of glucose over a time course of 360 hours. Error bars represent standard error (SE±) of the mean (n=2).
254
5.4.2.3 Analysis of lignin break-down products by HPLC-PDA-ESI-MSn
3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone
to 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone, and was observed in all
culture broth samples. However, the results of the analysis showed, that accumulation
of 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone varied again with
microorganism and carbon and energy source over a time course of 360 hours. In the Commented [AW[133]: Refer to figure
case of T. harzianum culture, accumulation of the product was only observed in the
presence of lignin and an extra carbon and energy source (Figure 5.12). The most rapid
rate of accumulation in culture broth with lignin as the sole carbon and energy source
for P. putida and P. saccharophila was observed between 18-72 hours and 18-48 hours,
respectively (Figure 5.13 A and B). In turn, P. fluorescens exhibited a different pattern of
accumulation. This strain started to accumulate the product only after 48 hours. The
most rapid rate of growth in culture broth supplemented with lignin as the sole carbon
and energy source for P. fluorescens was observed between 48-72 (Figure 5.13 C). The
highest concentration was detected in P. putida lignin supplemented broth at 96 hours
(0.045 mg/mL). In turn, the lowest level of accumulation of 3-hydroxy-1-(4-hydroxy-3-
methoxyphenyl)-1-propanone was observed in the case of P. fluorescens lignin
supplemented broth (maximum concentration at 360 hours: 0.026 mg/mL). Within P.
putida and P. saccharophila cultures, 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-
propanone content notably decreased after 96 and 144 hours, respectively. Thereafter,
the content remained stable until the end of fermentation. In turn, P. fluorescens grown
on lignin as the sole carbon and energy source showed a slow rate of increase in the
latter stage of fermentation (Figure 5.12 A). All bacterial strains exhibited very similar
patterns of of 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone accumulation in
the presence of glucose as an extra carbon and energy source (Figure 5.12 B/Figure 5.13
A, B and C). Inclusion of glucose generally increased the amount of 3-hydroxy-1-(4-
hydroxy-3-methoxyphenyl)-1-propanone in the case of P. fluorescens lignin
supplemented broth and resulted in a decrease in accumulation of the product in the
case of P. saccharophila culture (Figure 5.13 B and C). In comparison to bacterial species,
T. harzianum only started to accumulate 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-
255
propanone in the culture broth supplemented with lignin and glucose after 144 hours.
The highest rate of accumulation was observed up to 288 hours. Thereafter, the content
of 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone markedly reduced (Figure
5.13 D).
256
0.06 0.06
A B
Compound (mg/mL)
Compound (mg/mL)
0.04 0.04
PP PP
PS PS
0.02 0.02
PF PF
TH
0 0
0 100 200 300 400 0 100 200 300 400
Figure 4.1412: 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (GHP) content (mg/mL) in culture broths of P. putida, P. saccharophila, P. fluorescens and T.
harzianum strains supplemented with CIMV lignin (0.25 % w/v) alone (A) and in the presence of glucose (0.25 % w/v) (B). Error bars represent standard error (SE±) of the
mean (n=2).
257
0.06 0.06
A B
Compound (mg/mL)
Compound (mg/mL)
0.04 0.04
PP+lig PS+lig
0.02 0.02
0 0
0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
0.06
0.06
C
Compound (mg/mL)
D
0.04 Compound (mg/mL)
0.04
PF+lig
0.02 TH+lig+gluc
PF+lig+gluc 0.02
0
0 50 100 150 200 250 300 350 400 0
Time (hours) 0 50 100 150 200 250 300 350 400
Time (hours)
258
Figure 4.1513: 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone content (mg/mL) in culture broths of A) P. putida, B) P. saccharophila, C) P. fluorescens, D) T.
harzianum strains supplemented with CIMV lignin alone and in the presence of glucose over a time course of 360 hours. Error bars represent standard error (SE±) of the
mean (n=2).
259
3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone
to 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone, and was observed only
in bacterial broth samples. However, the result of the analysis showed, that
accumulation of 3-hydroxy-1-(4-hydroxy-3-dimethoxyphenyl)-1-propanone varied again
with microorganism and carbon and energy source over a time course of 360 hours Commented [AW[134]: Refer to figure
(Figure 5.14). The most rapid rate of accumulation in culture broth with lignin as the sole
carbon and energy source for P. putida and P. saccharophila, was observed between 48-
96 and 48-72 hours, respectively (Figure 5.15 A and B). In turn, P. fluorescens exhibited a
different pattern of accumulation. This strain started to accumulate product only after
144 hours. P. fluorescens lignin supplemented broth showed the most rapid rate of
accumulation between 144-216 hours of incubation (Figure 5.15 C). The highest
concentration was detected in P. saccharophila culture supplemented with lignin as the
sole carbon and energy source at 96 hours of incubation (0.020 mg/mL). In turn, the
lowest level of accumulation of 3-hydroxy-1-(4-hydroxy-3-dimethoxyphenyl)-1-
propanone was observed in the case of P. fluorescens lignin supplemented broth
(maximum concentration at 360 hours: (0.009 mg/mL). Within P. putida and P.
saccharophila cultures, 3-hydroxy-1-(4-hydroxy-3-dimethoxyphenyl)-1-propanone
content notably decreased by 216 hours and remained stable until the end of incubation.
In turn, P. fluorescens grown on lignin as the sole carbon and energy source showed a
slower rate of increase in the latter stage of fermentation (Figure 5.14 A). All strains
exhibited different patterns of 3-hydroxy-1-(4-hydroxy-3-dimethoxyphenyl)-1-
propanone accumulation in the presence of glucose as an extra carbon and energy
source (Figure 5.14 B). 3-hydroxy-1-(4-hydroxy-3-dimethoxyphenyl)-1-propanone
appeared more rapidly in these cultures, with the compound detected by 18 hours in P.
putida, 48 hours in P. fluorescens and by 48 hours in P. saccharophila cultures. Inclusion
of glucose notably decreased the level of accumulation of 3-hydroxy-1-(4-hydroxy-3,5-
dimethoxyphenyl)-1-propanone in the case of P. saccharophila culture and resulted in
higher levels in the case of P. putida and P. fluorescens cultures. The greatest rate of
increase for P. putida, P. saccharophila and P. fluorescens was observed between 8-72,
24-48, and 18-48 hours respectively. In P. putida cultures, level of product showed
260
marked decrease between 144-216 hour and thereafter remained relatively stable. The
general trend for P. saccharophila and P. fluorescens cultures showed, that after the
initial increase levels did not alter markedly up to the end of incubation period (Figure
5.15 A, B and C).
261
0.03 0.03
A B
Compound (mg/mL)
Compound (mg/mL)
0.02 0.02
PP PP
0.01 PS 0.01 PS
PF PF
0 0
0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
Figure 4.1614: 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone content (mg/mL) in culture broths of P. putida, P. saccharophila and P. fluorescens strains
supplemented with CIMV lignin (0.25 % w/v) alone (A) and in the presence of glucose (0.25 % w/v) (B). Error bars represent standard error (SE±) of the mean (n=2).
262
0.03 0.03
A B
Compound (mg/mL)
Compound (mg/mL)
0.02 0.02
PP+lig PS+lig
0.01 0.01
0 0
0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
0.03
Compound (mg/mL)
C
0.02
PF+lig
0.01
PF+lig+gluc
0
0 50 100 150 200 250 300 350 400
Time (hours)
Figure 4.1715: 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone content (mg/mL) in culture broths of A) P. putida, B) P. saccharophila, C) P. fluorescens strains
supplemented with CIMV lignin alone and in the presence of glucose over a time course of 360 hours. Error bars represent standard error (SE±) of the mean (n=2).
263
S(8-5)FA
Compound 5 produced an ion at m/z 401 in MS1 in negative mode which corresponds to
S(8-5)FA, and was observed only in bacterial broth samples. However, the results of the
analysis showed, that accumulation of S(8-5)FA varied again between microorganism
and carbon and energy source over a time course of 360 hours (Figure 5.16). The most
rapid rate of increase in S(8-5)FA content in P. putida, and P. saccharophila lignin
supplemented broths, was observed between 48-96 hours and 24-144 hours,
respectively. (Figure 5.17 A and B). Compared to the other species, P. fluorescens
exhibited a different pattern of accumulation. After a long lag period, a rapid rate of
increase in S(8-5)FA content was observed between 144-216 hours (Figure 5.17 C). The
highest concentration was detected in P. saccharophila culture, supplemented with
lignin as the sole carbon and energy source at 360 hours of incubation (0.015 mg/mL).
In turn, the lowest level of accumulation of S(8-5)FA was observed in the case of P. putida
lignin supplemented broth (maximum concentration at 360 hours: 0.010 mg/mL). S(8-
5)FA content slightly decreased in P. putida and P. saccharophila cultures between 144-
216 hours, and showed a general increasing trend for the remainder of the incubation.
In the case of P. fluorescens lignin supplemented broth, S(8-5)FA content remained
relatively stable between 216-288 hours, and subsequently increased during the final
72 hours of the incubation (Figure 5.16 A). All strains exhibited different patterns of S(8-
5)FA accumulation in the presence of glucose as an extra carbon and energy source
(Figure 5.16 B). Inclusion of glucose slightly decreased S(8-5)FA content in the case of P.
saccharohila and P. putida cultures, while it resulted in an earlier appearance of product
in P. fluorescens cultures compared with lignin only supplemented cultures. The greatest
rate of increase of S(8-5)FA for P. putida, P. saccharophila and P. fluorescens was
observed between 24-72, 8-96, and 18-96 hours respectively (Figure 5.17, A, B and C).
In P. putida culture, S(8-5)FA content markedly decreased by 216 hours and resulted in
a slow increase for the remainder of the incubation (Figure 5.17 A). The general trend for
P. saccharophila and P. fluorescens cultures showed, that following marked decrease
after 96 and 144 hours, respectively, S(8-5)FA content increased up to the end of the
incubation period (Figure 5.17 B and C).
264
0.02 0.016
A B
Compound (mg/mL)
0.016
Compound (mg/mL)
0.012
0.012
PP 0.008 PP
0.008
PS PS
0.004 PF 0.004 PF
0
0 50 100 150 200 250 300 350 400 0
0 50 100 150 200 250 300 350 400
Time (hours)
Time (hours)
Figure 4.1816: S(8-5)FA content (mg/mL) in culture broths of P. putida, P. saccharophila and P. fluorescens strains supplemented with CIMV lignin (0.25 % w/v) alone (A) and
in the presence of glucose (0.25 % w/v) (B). Error bars represent standard error (SE±) of the mean (n=2).
265
0.012 0.02
A B
Compound (mg/mL)
Compound (mg/mL)
0.016
0.008
0.012
PP+lig 0.008 PS+lig
0.004
0.004
0 0
0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
0.016
Compound (mg/mL)
C
0.012
0.008
PF+lig
0.004 PF+lig+gluc
0
0 50 100 150 200 250 300 350 400
Time (hours)
Figure 4.1917: S(8-5)FA content (mg/mL) in culture broths of A) P. putida, B) P. saccharophila, C) P. fluorescens strains supplemented with CIMV lignin alone and in the
presence of glucose over a time course of 360 hours. Error bars represent standard error (SE±) of the mean (n=2).
266
G(8-5)FA
Compound 6 produced an ion at m/z 371 in MS1 in negative mode which corresponds to G(8-
5)FA, and was observed in all culture broth samples. However, the results of the analysis
showed, that accumulation of G(8-5)FA varied again between microorganism and carbon
source over a time course of 360 hours. In the case of T. harzianum culture, accumulation of
the product was only observed in the presence of lignin and an extra carbon and energy source
(Figure 5.18). The most rapid rate of accumulation in culture broth with lignin as the sole
carbon and energy source for P. putida, P. saccharophila and P. fluorescens was observed
between 48-72, 24-48 and 24-72, respectively (Figure 5.19 A, B and C). The highest
concentration was detected in P. saccharophila culture, supplemented with lignin as the sole
carbon and energy source at 360 hours (0.022 mg/mL). In turn, the lowest level of
accumulation of G(8-5)FA was observed in the case of P. putida lignin supplemented broth
(maximum concentration at 144 hours: (0.015 mg/mL). Within P. putida and P. saccharophila
cultures, G(8-5)FA content slightly decreased between 144-216 hours Thereafter, the
compound increased at a slow rate till the end of the incubation period. In the case of P.
fluorescens lignin supplemented broth, the product accumulated at a lower rate up to the end
of the 360 hours incubation (Figure 5.18 A). All bacterial strains exhibited very similar patterns
of G(8-5)FA accumulation in the presence of glucose as an extra carbon and energy source
(Figure 5.18 B/Figure 5.19 A, B and C). Inclusion of glucose resulted in a marginal increase in
G(8-5)FA content in the case of P. fluorescens lignin supplemented broth (between 216-360 Commented [AW[135]: Earlier differences don’t look
significant
hours) and slightly lower levels of accumulation in P. putida and P. saccharophila culture broths
(between 48-216 hours and 96-360 hours, respectively) (Figure 5.19 A, B and C). In comparison
to bacterial species, T. harzianum only produced G(8-5)FA in the presence of glucose. The
product was first observed after 48 hours of incubation and generally continued to accumulate
till the end of the incubation period. (Figure 5.19 D).
267
0.03 0.03
A B
Compound (mg/mL)
Compound (mg/mL)
0.02 0.02
PP
PP
PS
0.01 PS
0.01 PF
PF
TH
0 0
0 100 200 300 400 0 100 200 300 400
Figure 4.2018: G(8-5)FA content (mg/mL) in culture broths of P. putida, P. saccharophila and P. fluorescens and T. harzianum strains supplemented with CIMV lignin (0.25 %
w/v) alone (A) and in the presence of glucose (0.25 % w/v) (B). Error bars represent standard error (SE±) of the mean (n=2).
268
0.03 0.03
Compound (mg/mL)
Compound (mg/mL)
A B
0.02 0.02
PP+ lig PS+lig

0.01
0
0 100 200 300 400 0
time 0 100 200 300 400
time
0.03 0.004
Compound (mg/mL)
Compound (mg/mL)
C 0.003 D
0.02
0.002
PF+lig
0.01 TH+lig+gluc
PF+lig+gluc 0.001
0 0
0 100 200 300 400 0 100 200 300 400
time
time
Figure 4.2119: G(8-5)FA content (mg/mL) in culture broths of A) P. putida, B) P. saccharophila, C) P. fluorescens, D) T. harzianum strains supplemented with CIMV lignin alone
and in the presence of glucose over a time course of 360 hours. Error bars represent standard error (SE±) of the mean (n=2).
269
5.5 DISCUSSION
Better knowledge of microbial biodiversity is a key to greater understanding of lignin

degradation (Datta et al., 2017). Lignin-degrading microorganisms especially fungi, have
already revealed a big interest as potential biomass degraders for large-scale
applications due to their ability to produce an array of different extracellular enzymes
(Dashtban et al., 2010). Additionally, lignin-degrading bacteria also represent an almost
inexhaustible source for different industrial enzymes and offer advantages over other
sources due to their rapid growth and reasonable production costs (Ghribi et al., 2016).
However, induction of ligninolytic enzymes highly depends on specific strains and
cultivation conditions (Asina et al., 2017). Hence, discoveries of novel isolates and more
detailed insights into their ligninolytic capabilities in different environments could help
to identify new effluent streams of aromatics for use in value-added products (Abdelaziz
et al., 2016).
In this study, T. harzianum, P. putida, P. saccharophila and P. fluorescens were examined

in detail for growth in M9 and YNB minimal media supplemented with 0.25 % (w/v) of
lignin. Based on preliminary results presented in Chapter 4 it was postulated, that fungal
and bacterial growth in liquid culture supplemented with a low amount of lignin as the
sole carbon and energy source would be a practical option in the context of further
analysis of lignin break-down products or enzyme purification. All the test samples were
compared to positive control samples, where glucose was added to the media in order
to see the effect of inclusion of a readily utilisable carbon and energy source on lignin
metabolism. The results confirmed, that tested T. harzianum strain is capable of utilizing
lignin as the sole carbon and energy source for its growth. The lack of growth observed
in the first 48 hours of incubation, showed a long lag phase of this strain in YNB even
with inclusion of glucose. In general, poor growth of T. harzianum in YNB liquid media
supplemented with CIMV lignin could be due to the limited ability of this strain to
depolymerize lignin polymer in a minimal media environment. It was observed, that
addition of a readily utilisable carbon and energy source markedly increased the extent
of growth, especially in the latter stage of the incubation (Table 5.1). Some studies have
already reported, that fungi can break down lignin but glucose has to be added as an
270
extra carbon and energy source. Degradation of lignin by P. versicolor and two other
wood-rotting fungi could not be achieved without the presence of glucose (Day et al.,
1952; Kirk et al., 1976). Another explanation could be a low tolerance of fungi to liquid
media. Results from initial experiments reported in Chapter 4 showed, that a solid-based
environment and a bigger surface accessibility was much more preferable for the growth
of the fungal strain here investigated.
In the case of bacterial species investigated here, this study confirmed the ability to
utilize lignin as the sole carbon and energy source for their growth as observed previously
(Chapter 4). However, inclusion of glucose as an additional utilisable carbon and energy
source markedly altered the growth patterns over a time course of 360 hours. The
maximum OD590 reached by cultures of the Pelomonas and Pseudomonas sp. in M9
media supplemented with CIMV lignin, was two to three times lower than the level
reached in the presence of CIMV lignin and extra carbon and energy source (Figure 5.4 A
and B). Rapid growth in the first 24 hours in M9 media supplemented with CIMV lignin
and glucose was most likely related to metabolising glucose., which was most Commented [AW[136]: NB IN RESULTS -NEED TO MAKE SURE
YOU SAY CELLS GO INTO STATIONANRY PHASE AFTER 24 ON
presumably completely utilised within this time frame. It is interesting, that all bacterial GLUCOSE- REMOVE COMMENTS ABOUT CELL DEATH IN RESULTS-
ALSO WORTH HIGHLIGHTING IN RESULTS THAT IN CONTRAST TO

strains remained in stationary phase for the remainder of the incubation, with the CELLS ON GLU AND LIGNIN- CELLS GROWN ON LIGNIN ALONE DID
NOT REACH STATIONARY PHASE OVER 360 HOURS INCUBATION
exception of P. saccharophila which showed an increase in OD between 288-360 hours.
ALSO SOME INDICATON THAT P.SACC ON GLU SHOWED EVIDENCE
This could suggest, that this strain switched to different metabolic pathway (Figure 5.4 OF GROWTH BETWEEN 288 AND 360 HOURS
A). In general, the lack of death phase in media supplemented with CIMV lignin as the
sole carbon and energy source indicated that lignin was further metabolised suggesting,
that longer incubation times could be considered for higher growth (Figure 5.4 B). Similar
findings were previously reported by Zhu et al., (2017).Their studies showed, that B. Commented [AW[137]: Interesting paper – very relevant to
your study!
ligninophilus was able to utilize alkaline lignin as the sole carbon and energy source.
Additionally, when the same strain was grown in media supplemented with lignin and
glucose, cultures showed continuous growth after glucose was exhausted Commented [AW[138]: Had a look at paper and I think this is
what they were saying
demonstrating, that the lignin provided a carbon and energy source to support the
growth in these cultures.
The ligninolytic capability of the microbial strains was further investigated by testing
spent culture media taken at different time intervals for the presence of lignin
degradation products. According to the literature, low molecular weight compounds
271
released from lignin show wide variation due to the diversity of lignin chemical
structures and enzymatic strategies of lignin depolymerization, (Fisher and Fong, 2014).
In this study, analysis of cell-free extracts revealed the presence of a range of phenolics
including the following compounds: ferulic acid, coumaric acid, 4-0-5’-coupled diferulic
acid, 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone,3-hydroxy-1-(4-hydroxy-
3,5-dimethoxyphenyl)-1-propanone, S(8-5)FA and G (8-5)FA) (Table 5.2/Figure 5.5). The
presence of ferulic acid, coumaric acid and 4-0-5’-coupled diferulic acid in all samples
has shed light on the pathways utilised by these strains to degrade CIMV lignin which is
derived from wheat straw. Hydroxycinnamic acids are present in much higher amounts
in grass than in wood and form cross-links between lignin and hemicellulose (Herring et
al., 2016). Coumaric acid is linked to lignin by ester bonds, ferulic acid by both ether and
ester bonds (Al Arni et al., 2007) and 4-0-5’-coupled diferulic acid by ester bonds
(Klepacka, J., 2006). Observation of coumaric acid, ferulic acid and 4-0-5’-coupled
diferulic acid in spent culture media, indicated the presence of esterases. These enzymes
are active on the ester bonds of hydroxycinnamic acids (Herring et al., 2016) and have
been already identified in bacteria (including P. fluorescens) and some fungal strains
(Bartolomé et al., 1997). Additionally, the presence of etherases could also be a factor
(Flores, 2013). Accumulation of all three hydroxycinnamic acid products was observed
from the early stages of the incubations indicating, that esterases were rapidly expressed
in these cultures. It is very likely, that some ester/ether linkages were already broken
prior to microbiological degradation (during the organosolv or sterilisation process).
Cleavage of both types of bonds has previously been observed during acid-catalysed
organososlv (Rana and Rana, 2017) but more research would need to be carried out to
fully confirm this theory.
Following identification of lignin-related phenolics described above, focus was given to

core lignin break-down products. The presence of 3-hydroxy-1-(4-hydroxy-3-
methoxyphenyl)-1-propanone and 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-
propanone indicated cleavage of β-O-4 ether linkages as a part of the β-O-4 ether
catabolic pathway (Reiter et al., 2013). β-aryl ether bonds account for 50–70 % of all
inter-unit linkages in lignin. Since cleavage of β-aryl ether bonds is the most important
process in lignin degradation, many studies have focussed on characterization of β-aryl
272
ether catabolism to clarify the mechanism of lignin biodegradation (Helmich et al., 2016). Commented [AW[139]: This is not being done in these
experiments – need to be careful not to suggest so
β-O-4 ether pathway studied in bacterial species revealed, that β-O-4 ether linkages are
cleaved via multiple enzyme reactions. Additionally, enzymes in the cleavage of β-O-4
ether linkages have been considered to show substantial activity only on dimeric lignin-
model synthetic compounds, with low activity on polymeric substrates (Ohta et al.,
2015). Hence, it is very likely that CIMV lignin used in this study was already partially
depolymerised. During the organosolv process, β-aryl ether linkages are among the first
bonding types to be cleaved (Reiter et al., 2013). Moreover, subjecting lignin to extra
chemical changes during sterilisation process could enhance bacterial capability to
metabolise the substrate. Cleavage of β-O-4 ether linkages present in partially
depolymerized, low-molecular-weight lignin using intracellular enzymes was previously
reported in the literature (Ohta et al., 2017). However, further characterisation of CIMV
lignin would need to be carried out to fully confirm this theory. So far it has been
determined, that β-O-4 pathway involves an enzymatic cascade catalysing the reductive
cleavage of the ether bond and the oxidation of the secondary alcohol via reduction of
NAD+ and oxidation of two molecules of reduced glutathione (Pollegioni et al., 2015).
Initial oxidation of the a-hydroxyl group to the corresponding ketone is catalysed by the
NAD- dependent dehydrogenase (Bugg et al., 2011b). The β-O-4 ether linkages between
monoaromatic units are then cleaved by β-etherases belonging to the glutathione-S-
transferase (GST) superfamily. In the last step, glutathione is removed from the
intermediate compounds by GSTs with β-glutathione thioetherase. Based on this, it is
proposed that 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone, also known as
guaiacyl hydroxyl propanone (GHP), and 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-
1-propanone, a methoxylated derivative of GHP commonly known as syringyl hydroxyl
propanone (SHP), are an end metabolites of a three-stage process (Bugg et al., 2011a;
Reiter et al., 2013; Ohta et al., 2015). Cleavage of β-O-4 ether linkages in lignin model
compounds by glutathione-S-transferases as a part of β-O-4 ether degradation pathway
has previously been reported in Pseudomonas acidovorans (Bugg et al., 2011b).
However, no evidence suggesting presence of these type of enzymes in Trichoderma sp.
has been found yet.
273
The compounds such as S(8-5)FA and G(8-5)FA clearly indicated the presence of 8-5
linkages with the core lignin structure. So far it has been recognised, that break-down of
the phenylcoumaran component of lignin can be initiated by the oxidation of the
sidechain, then oxidation of the heterocyclic ring and finally oxidative Ca–Cb bond
cleavage (Bugg et al., 2011b). S(8-5)FA and G(8-5)FA are recognised products of the
second step in the phenylcoumaran catabolic pathway, which involves the
dehydrogenation step catalysed by an aryl alcohol dehydrogenase (ADH) and subsequent
oxidation by a benzaldehyde dehydrogenase. These enzymes have been previously
identified in P. putida (Kamimura et al., 2017) and benzaldehyde dehydrogenase was
characterised by Zahniser et al., 2017. It is of interest, that in this study evidence was
observed for the presence of phenylcoumaran benzylic ether reductase (PCBER) in P.
putida and P. saccharophila cultures, which reduce phenylpropanoid dimers (Niculaes et
al., 2014). Reduced G(8-5)FA products were tentatively idenfied in these samples.
The lignin break-down products identified here clearly indicated a range of different
mechanisms involved in lignin degradation. The different microbial species showed
different patterns of accumulation and while there were insufficient representative
samples to carry out statistical analysis due to logistical reasons, clear trends were
apparent. Comprehensive characterisation of lignin degradation patterns showed, that Commented [AW[140]: I think you need to say something
along these lines to cover lack of statistics- it would have been
ligninolytic capability of T. harzianum differed from bacteria. Analysis of fungal broth logistically impossible to include more reps-
samples revealed lower levels of ferulic and 4-0-5’ diferulic acid compared to bacterial Commented [AW[141]: Putida accumulated lowest coumaric
cultures (Figures 5.9 and 5.11). In contrast with the bacterial species (P. putida, P.
saccharophila and P. fluorescens), T. harzianum did not show the ability to metabolize
coumaric acid (Figure 5.7) with accumulated levels persisting throughout the incubation
period. Lower levels of 4-0-5’ diferulic acid could indicate, that fungal esterase had a
poor affinity for this substrate. In addition, no core lignin break-down products were
identified in M9 media supplemented with CIMV lignin as the sole carbon and energy
source. Only 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone and very low
levels of G (8-5) FA were observed in the presence of lignin and glucose as an extra
carbon and energy source (Figures 5.13 and 5.19). This indicated, that metabolism of the
core lignin structure was minimal with this species. Analysis of results for the bacterial
strains showed, that P. putida and P. saccharophila appear to use similar pathways for
274
lignin metabolism. In turn, lignin degradation by the P. fluorescens strain differed from
the rest of species (Figures 5.9, 5.11, 5.13, 5.15, 5.17 and 5.19). In general, P. putida and
P. saccharophila produced the highest amount of lignin break-down products ( ferulic Commented [AW[142]: For most compounds differences did
not look significantly different and generally higher than fluoresens
acid, 4-0-5’-coupled diferulic acid, 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl) -1- and this fits with previous sentences
propanone, 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl) -1-propanone and G(8-5)FA)

(Figures 5.9, 5.11, 5.13, 5.15 and 5.19) . The exceptions were coumaric acid and S(8-5)FA
which showed considerably lower levels in P. putida cultures (Figures 5.7 and 5.17).
Coumaric acid content in particular, was markedly lower in these cultures indicating that
this compound is readily catabolized by this species. The lowest accumulation of
compounds among bacterial species was observed in the case of lignin degradation by
P. fluorescens (ferulic acid, 4-0-5’-coupled diferulic acid, 3-hydroxy-1-(4-hydroxy-3-
methoxyphenyl)-1-propanone and 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-
propanone). There were also longer lag periods before the appearance of products
particularly with lignin as the sole carbon and energy source (Figures 5.9, 5.11 5.13 and
5.15). For all microbial strains, the amount of compounds accumulated in the first 24
hours was generally low. The biggest increase was mostly observed between 24-144
hours, with the maximum amount of product in a time range of 96-144 hours. It is also
of interest, that inclusion of glucose resulted in a marked increase in the amount of core
lignin break-down products produced by P. fluorescens and T. harzianum and in general Commented [AW[143]: Differences for HCAs do not appear
significant
there was a clear decrease in the accumulation of compounds produced by P. putida and
P. saccharophila (Figures 5.7, 5.9, 5.11, 5.13, 5.15, 5.17 and 5.19). Findings described
above corroborate growth patterns obsesrved for T. harzianum which showed no growth
during the first 24 hours of incubation, poor growth in the presence of CIMV lignin, and
increased growth in the presence of an extra carbon and energy source. The higher levels
of lignin degradation compounds produced by Pelomonas and Pseudomonas sp. in the
presence of lignin and glucose could be explained by rapid growth in media
supplemented with extra carbon and energy source. In turn, the low content of lignin
break-down compounds observed with P. fluorescens may be related to the slightly
lower levels of growth in these cultures. P. fluorescens cultures showed lower OD590
values and may indicate less efficient utilization of organosolv lignin by this strain.
Differences observed in the patterns of lignin degradation with the different
microorganisms were in good agreement with data published in the literature. It has
275
been postulated, that the heterogeneous nature of lignin limits the catabolic
compatibility of lignin-degrading microbes with the type of lignin source. This means,
that not all lignin-degrading organisms can utilize all types of lignin sources and their
derivatives (Abdelaziz et al., 2016).
Based on the results presented above, it is clear that lignin degradation is a multi-
enzymatic process and its break-down produces a wide range of aromatic compounds.
These processes also create low molecular weight lignin oligomer with new functional
groups e.g. hydroxyl and carboxylic groups in the case of esterases and hydroxyl and
ketone groups in the case of etherases. Such modifications may improve properties for Commented [AW[144]: I think it is important to point out that
you can get products which can be used for resins/polyurethanes
use in resins/polyurethanes. Moreover, through the natural variety in microbial
metabolic products and the possibilities of metabolic engineering, there exist many
routes to convert low molecular weight lignin molecules to value-added end-products
(Abdelaziz et al., 2016). The relatively high yields of ferulic acid and 3-hydroxy-1-(4-
hydroxy-3-methoxyphenyl)-1-propanone (GHP) produced by P. saccharophila, could be
particularly significant in a context of synthesis of new bio-based products. Ferulic acid
is in its self of interest for reported anti-cancer properties and its potential as a
cardiovascular drug or skin care product (Srinivasan et al., 2007). In addition, this
compound can also be used for synthesis of vanillin and PCA. According to Rosazza et al.,
(1995), production of potential biopolymers derived from ferulic acid is also possible.
Synthesis of poly(carbonate-amide)s and poly(anhydride ester) was recently described
by Llevot et al., (2016). In turn, 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone
(GHP), could most likely become the platform aromatic chemical for sustainable
industries. A bisphenol synthetized from GHP would potentially be used as a raw material
for epoxy resins, as a hardener for epoxy and urethane resins and as a raw material for
new functional polyesters and polycarbonate. Moreover, coniferyl alcohol generated
from GHP could be a valuable source of compounds used as medicines for cancers and
diabetes; functional foods and cosmetics with antioxidizing activity (Ohta et al., 2017).
Other phenolic products may also have industrial applications. It has been already
postulated, that lignin-related phenolics such as coumaric acid and 4-0-5’-coupled
diferulic acid could have good potential as antioxidants in the food industry. P-coumaric
acid is additionally recognized as a chemical with potential for the pharmaceutical
276
industry as a drug against stomach cancer (Al Arni et al., 2007). Applications for
oligolignols such as S(8-5)FA and G(8-5)FA have not yet been identified , but it has been
suggested, that they could also serve as antioxidants (Dima et al., 2015). However it must
be noted, that quantities produced in this study were low and for these products to be
utilized in industry, they would need to be produced in sufficient yields to be
economically viable. Commented [AW[145]: I think you need to add this proviso
This study provided an insight into the mechanisms of microbial-mediated lignin break-
down, suggesting that P. saccharophila and possibly P. putida could be a promising
microorganism for bioconversion of lignin. In order to completely understand the
metabolic characteristics of lignin degradation and identify enzymes involved in the
decomposition process, more experiments would need to be carried out in future work.
Apart of from the proposed enzymes and pathways, there may be still a range of
additional catabolic pathways and enzymes involved (Fisher and Fong, 2014). Studies
carried out by Kawai et al., (1999) showed, that β-O-4 ether linkages can be cleaved by
laccase. Moreover, Ahmad, (2010) recently observed, that cleavage of β-O-4 ether can
be carried out in the presence of a bacterial novel manganese-dependent lignin
peroxidase, DypB which has been identified in P. fluorescens species. Hence, further
study to determine the genetic and enzymatic basis of the identified break-down
compounds could help to engineer new routes for production of lignin-derived chemicals
from renewable feedstocks (Sainsbury et al., 2013). It has been proposed, that a deeper
knowledge of lignin-derivatives interaction with lignin-degrading enzymes is another,
important step in the development of lignocellulosic biorefineries for the 21st century
(Bugg et al., 2011b).
277
5.6 CONCLUSIONS
T. harzianum, P. putida, P. saccharophila and P. fluorescens were examined in detail for

their ability to grow in liquid media supplemented with CIMV organosolv lignin and to
produce lignin degradation products. The results of the growth experiment confirmed,
that all strains have the ability to utilize CIMV lignin as the sole carbon and energy source.
Analysis of cell-free extracts revealed a range of core lignin break-down products and
lignin-related phenolics. Identified compounds clearly indicated a different mechanisms
involved in lignin degradation, including cleavage of ester and β-O-4 ether linkages and
catabolism of phenylcoumarans. Characterisation of lignin degradation patterns showed,
that ligninolytic capability of fungal strain differed from bacteria. Studied fungus showed
poor ability to utilise CIMV lignin in the absence of an extra carbon and energy source.
Although, all bacterial species were able to use lignin as the sole carbon and energy
source, adding glucose markedly increased the rate and extent growth, and in case of P.
fluorescens cultures also resulted in increased accumulation of core lignin break-down
products. In conclusion, it is proposed, that P. saccharophila could be a promising
organism for bioconversion of CIMV lignin and sustainable production of bio-based
products, including vanillin, PCA and polyesters. Additional products such as GHP may
also be a source of chemicals for the food and medical industry as well as building block
for the production of polyesters, polycarbonates or epoxy and urethane resins.
278
Chapter 6
279
Chapter 6: CHARACTERISATION OF LIGNINOLYTIC CAPABILITY OF P.
SACCHAROPHILA- A PROMISING CANDIDATE FOR
BIOCONVERSION OF ORGANOSOLV LIGNIN
6.1 ABSTRACT
Ligninolytic capability of P. saccharophila was studied for its potential application in

biorefining processes. The main aim of this chapter was to determine the enzymatic basis
of P. saccharophila-mediated organosolv lignin metabolism. P. saccharophila, was grown
over 144 hours in M9 liquid media supplemented with 0.25 % (w/v) of CIMV organosolv
lignin as the sole carbon and energy source. Extracellular and intracellular proteins
extracted from culture media were purified and quantified based on the Bradford assay.
The protein profile and molecular weight distribution was further studied by SDS-PAGE
analysis. Inclusion of copper to the lignin supplemented media was also investigated for
its effect on expression of ligninolytic enzymes, specifically laccases. Additionally, lignin
samples were recovered from fermentation broth and characterized by SEC and 31 P-
NMR. The results of the analysis revealed, that intracellular proteins increased steadily
over the time course reaching a maximum at 144 hours and exhibited the highest
intensity bands at 96 hours when analysed by SDS-PAGE. Extracellular proteins were only
above detection limit in the 96 hours sample, which also gave rise to faint bands in an
SDS-Page gel. Protein gels showed multiple bands in the region between 35-70 kDa, and
laccases and Dyp-type peroxidases are reported to be in this size-range. The activity of
laccase was observed in both intracellular and extracellular samples in phosphate buffer
at pH 6. The addition of CuSO4 to the media resulted in the production of extracellular
laccase by 48 hours of incubation and increased the amount of intracellular laccase at
144 hours. Analysis of lignin samples by SEC and 31P-NMR revealed, that the P.
saccharophila strain modified lignin fractions. Overall results confirmed, that P.
saccharophila is a promising candidate for production of high-value chemicals and other
bio-based products.
Keywords: • P. saccharophila • CIMV lignin • Ligninolytic enzymes • Laccase • Lignin

bioconversion
280
6.2 INTRODUCTION
The key challenge for the conversion of lignin into high-value products is the recalcitrant
and complex nature of the lignin polymer (Gall et al., 2017). As discussed previously,
bioconversion of lignin with microbes offers an alternative lignin valorisation approach
with good potential (Xie et al., 2016).
Despite the fact that all the basic and applied research work have mostly focused on
fungi, their use in industrial applications is not very practical. This is because of the
structural hindrance caused by fungal filaments, requirement of a long lag period and
thus very slow degradation of lignin. In addition, they have a high demand for particular
culture conditions such as humidity, aeration, temperature and pH which are not
compatible with industrial processing environments (Patil, 2014). The development of
fungal biocatalysts for large-scale lignin modification is also hampered by the challenges
of fungal protein expression (Bugg and Rahmanpour, 2015). Therefore, in recent years
there has been a growing interest in the identification of new- lignin degrading bacteria
(de Gonzalo et al., 2016).
Bacteria have unique advantages over fungi, including their remarkable resilience in
varied environmental conditions, rapid growth rates and ease of genetic manipulation
(Tian et al., 2016). They are able to metabolize lignin to provide sufficient carbon and
energy for growth and lead to more efficient lignin bioconversion within a short period
of time (Asina et al., 2017).
So far, lignin degradation has been already investigated using several bacterial strains
including Rhodococcus jostii, Streptomyces viridosporus, Sphingobium sp., Nocardia sp.,
Pseudomonas sp., Comamonas sp. or Bacillus sp. (Zhu et al., 2017). Many studies suggest,
that Pseudomonas sp. are the most efficient lignin-degrading bacteria (Lotfi, 2014). The
ability of P. putida, P. fluorescens, P. paucimobilis or P. aeruginosa to produce a range of
ligninolytic enzymes including laccase, vanillin dehydrogenase and dye decolorizing
peroxidase has already been reported in the literature (Kuddus et al., 2013; Arunkumar
et al., 2014; Ding et al., 2015; Lotfi, 2014; Santos et al., 2014). However, no evidence
indicating bioconversion of lignin by P. saccharophila and expression of ligninolytic
enzymes has been reported to date.
281
As presented in Chapter 5, P. saccharophila showed a large capacity for bioconversion of
organosolv CIMV lignin. Lignin degradation products, clearly indicated expression of a
range of different enzymes involved in this process. Hence it was proposed that
ligninolytic enzymes should be expressed, purified, and identified to assess which lignin
bonds are actually being cleaved. The main aim of the study presented in this Chapter
was to determine the enzymatic basis of P. saccharophila-mediated organosolv lignin
conversion. As high yields of break-down products produced by this strain indicated its
potential for synthesis of high-value chemicals and biomaterials it was proposed, that
monitoring the activity of ligninolytic enzymes with lignin substrate may shed new light
on the development of lignin bioconversion technologies. P. saccharophila was selected
from a range of bacterial species which were previously investigated for their ligninolytic
capability (Chapter 5). This strain was grown for 144 hours in M9 liquid media
supplemented with 0.25 % (w/v) CIMV lignin. Extracellular and intracellular enzymes
were extracted from liquid cultures, purified and quantified using the Bradford assay.
The protein profile and molecular weight distribution was further studied by Sodium
Dodecyl Sulphate - Polyacrylamide Electrophoresis (SDS-PAGE) analysis. The effect of
inclusion of copper in the media on ligninolytic enzyme expression was also investigated.
Additionally, lignin samples were recovered from fermentation broth and characterized
by Size Exclusion Chromatography (SEC) and Phosphorus-31 Nuclear Magnetic
Resonance (31P-NMR). Results showing the ability of P. saccharophila to modify and
degrade CIMV lignin were discussed in a context of its future use in biorefining.
282
described below. The procedures used for growth experiment were carried out under
aseptic conditions, using sterile chemicals and laboratory equipment. The reagents for
molecular analysis were of molecular biology grade. Unless otherwise stated, all plastic
ware and commonly used chemicals of the highest grade available were purchased from
Fisher Scientific (Lecicestershire, UK) or Sigma-Aldrich Company Ltd (Dorset, UK). Any
glassware used in the carried out procedures were of Duran or Pyrex brand. For all steps,
purified water, (Elga Purelab Classic UV, VWS UK Ltd, 18.2 mega ohm-cm resistivity at 25
°C) was used unless otherwise stated.
6.3.1 FEEDSTOCKS
The feedstock used in this study was Compagnie Industrielle de la Matière végétale
(CIMV) organosolv wheat straw lignin as a standard, which was kindly supplied by CIMV Commented [AW[146]: In full first time in methods-
Company, Pomacle, Champagnes-Ardennes, France.
Bacteria used in this study was: Pelomonas saccharophila (NCIMB 8570; coded PS). It was
supplied in glycerol stock by IBERS, Aberystwyth University, Wales, UK.
6.3.3 CHARACTERISATION OF LIGNIN METABOLISING BACTERIA

6.3.3.1 Growth of P. saccharophila in M9 liquid media supplemented with CIMV lignin
P. saccharophila cultures were grown in duplicate, in 250 mL shake flasks in 100 mL of
M9 minimal media buffered to pH7 (prepared as described in section 4.3.5.2) (pH 7
optimal for bacterial growth) and supplemented with CIMV lignin as the sole carbon and
energy source. In order to avoid complexing of lignin with other media components
during autoclaving, CIMV lignin was freeze-dried, autoclaved and added separately to
sterile media in the following amount: 0.25 % (w/v). Single colonies from LB agar plates
(prepared as described in section 4.3.3) were inoculated into 10 mL of fresh LB liquid
media, incubated at 30 oC with shaking at 180 rpm and grown to an OD 590 = 0.75 for use
as an inoculum. M9 liquid media supplemented with lignin, was inoculated with 2 mL of
283
the inoculum culture. Cultures were continuously mixed throughout the incubation
period on a rotary shaker at 180 rpm at 30 oC for 144 hours. Aliquots of each culture
(1mL) were removed after 24, 48, 72, 96 and 144 hours and the OD590 was monitored
using a CO8000 cell density meter (Biochrom, Wpa, Cambridge, UK). All readings were
taken in triplicate and the cultures exhibiting readings above 1.0 OD590 were diluted 2-5-
fold in the same culture media. Two control treatments were also included, a positive
control with microorganisms grown in media supplemented with 0.25 % (w/v) of glucose
and a negative control with media without carbon and energy source. To investigate the
effect of copper, 0.25mM of CuSO4 were added to the media, inoculated with P.
saccarophila and incubated in conditions described above. Aliquots of each culture (1mL)
were removed after 48, 72 and 144 hours.
6.3.4 ENZYME ASSAYS

6.3.4.1 Laccase activity of extracellular and intracellular extracts of P. saccharophila
cultures
Enzymes produced by P. saccharophila were assayed for laccase activity following a
method adapted from Nitheranont et al., (2011). The assay mixture was prepared in a
96-well microliter plate and consisted of 190 μL of master mix including: McIlvaine
buffer (0.2M disodium hydrogen phosphate, 0.1 M citric acid)(McIlvaine, 1921), 1 mM
ABTS solution, 1 mM CuCl2 and 10 μL of enzyme extract to make up to 200 μL. 190 μL of
the master mix solution was added to 10 μL of enzyme extract, covered with a plate
sealer sheet (Genetix, Ltd, New Milton, UK) and incubated for 14 hours at 30 oC.
Oxidation of ABTS was analysed by monitoring the increase in absorbance at 420 nm (ε
= 3.6 × 104 M–1 cm –1). One unit of enzyme (U) was defined as the amount of enzyme
required to oxidize 1 μmol of substrate per minute at 30 oC. The assay was carried out
with duplicate replicates. For the positive control, enzyme extract was replaced with 10
μL of 0.025 mg/mL laccase from Trametetes versiciolor, and 50 mM Tris-HCL buffer
replaced the enzyme extract in the negative control. Later assays were carried out
replacing McIlvaine buffer with 0.1 mM phosphate buffer at pH 6. The optimal
concentration was chosen based on a series of 5 different dilutions carried out prior to
experiments.
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6.3.5 PROTEIN PURIFICATION AND EXPRESSION
6.3.5.1 Ammonium sulphate precipitation for extracellular enzymes
Ammonium sulphate precipitation was carried out as described in section 4.3.11.1.
6.3.5.2 Desalting proteins

Desalting of proteins was carried out as as described in section 4.3.11.2.
6.3.5.3 Preparation of cell-free extracts

Preparation of cell-free extracts was carried out according to the procedure of Fisher
Scientific . 0.5 mL of cell lysis buffer consisting of 50 mM Tris-HCl at pH 7, with protease
inhibitors (1000 units/mL trasylol, 0.2M phenylmethylsulfonylfluoride (PMSF), 0.1M Commented [JG[147]: Which inhibitors
1,10 phenantroline, 0.1M benzamidine) and 1 mg/mL of lysozyme was added to tubes
with pelleted bacterial cells (kept on ice) and mixed well by vortexing for 10 seconds.
Following this, 5 μL of 1M MgCl2 and 5 μL of 1 mg/mL of DNAse was added, and mixed
well by vortexing for 10 seconds to improve lysis and reduce viscosity caused by DNA
content. The mixture was incubated on ice for 15 minutes and sonicated 3 times for 10
seconds with 30 sec intervals. The procedure was repeated 3 times, with at least 5
minutes rest on ice between each run. The lysate was centrifuged at 10 000 x g for 30
minutes at 4 oC, the cell free extract was aliquoted and stored at -80 oC for further
analysis.
6.3.5.4 Determination of protein concentration

Protein concentration was estimated using the BioRad Protein Assay (Hemel,
Hempstead, UK) based on the method of Bradford (Bradford, 1976). The assay involved
addition of 40 μL of crude extracellular extract and/or 4 μL of of crude cell-free extract
to 1 mL of 1x Bradford reagent in a semi micro cuvette and incubation at room
temperature for 20 minutes. Acidic dye (Coomassie Brilliant Blue G-250) included in the
Bradford reagent, created a calorimetric complex with a protein solution and its
absorbance was measured using a spectrophotometer at 595nm. A standard curve
(Figure 6.1) was generated using series of dilutions of Bovine Serum Albumin (BSA) from
New England Biolabs (Ipswich, MA, USA) added to 1 mL of 1x Bradford reagent. The
amount of protein in each enzyme fraction was then determined from the calibration
plot below.
285
0.6
0.5
Absorbance at 590nm
0.4
y = 0.0481x + 0.0877
0.3
R² = 0.9933
0.2
0.1
0
0 2 4 6 8 10 12
BSA concentration (µg)
Figure 4.221: Bovine serum albumin (BSA) protein standard curve for the BioRad protein assay.
6.3.5.5 Protein expression
6.3.5.5.1 SDS-PAGE samples preparation

Extracellular and intracellular proteins samples were thawed on ice. 50 μL of the
supernatant was removed from each sample and transferred to a new centrifuge tube.
An equivalent amount of SDS-sample buffer composed of 50 mM Tris-HCl, (pH 7.0), 5 %
(w/v) SDS, 25 % (v/v) glycerol and 0.002 % (w/v) bromophenol blue was added to the
centrifuge tube. After heating over a boiling water bath for 5 minutes, samples were
applied in the well of the stacking gel for electrophoresis.
6.3.5.5.2 SDS-PAGE gel preparation

SDS-PAGE employs gels composed of a stacking gel with large pores and a separating gel
with smaller pores of a size suitable for separation of proteins of interest. The stacking
gel is prepared within a relatively low pH (6.8) and ionic strength buffer, whilst the
separating gel is at a relatively high pH (8.5) and ionic strength buffer. Two buffer systems
provide a discontinuous pH and voltage gradients in the gel, allowing the SDS denatured-
proteins to be concentrated in the stacking gel and then separated according to their
sizes during electrophoresis in the running gel. Proteins extracts were separated on 12
% acrylamide gels (composition shown in Table 6.1). The gels were cast using a Bio-Rad
electrophoresis apparatus. Electrophoresis was conducted using a tank buffer composed
of 25 mM Trizma base, 192 mM glycine and 0.1 % (w/v) SDS. SDS-denatured protein
286
sample was loaded into each well of the gel. Electrophoresis was performed at a fixed
current of 30 mA and under a variable voltage, until the bromophenol blue had reached
a few mm to the base for 1.5 to 2.5 hours.
Table 4.151: Solutions used for preparation of a mini 12 % SDS-polyacrylamide gel.
Gel system 30 % (w/v) Buffer volume 20 % (w/v) TEMED (μL)

polyacrylamide (mL) (mL) H2O (mL) APS (μL)
Running gel 3.6 RGB 2.25 3.15 20 15

(12%)
Stacking gel 0.75 SGB 1.25 3 20 15

(4.5%)
Stock RGB, (running gel buffer) 1.5 M Tris-HCl (pH 8.5), 0.4 % (w/v) SDS
Stock SGB, (stacking gel buffer) 0.5 M Tris-HCl (pH 6.8), 0.4 % (w/v) SDS
APS, freshly prepared ammonium persulphate
TEMED, N, N, N,’N’-tetramethylethylenediamine
6.3.5.5.3 Detection of protein

Post electrophoresis, gels were stained for protein overnight with 50 mL of 0.25 % (w/v)
Coomassie Blue R-250 dissolved in 50 % (v/v) methanol and 10 % (v/v) acetic acid. The
gels were then briefly washed with water and destained with 50 mL destaining solution
(30 % (v/v) ethanol and 10 % (v/v) acetic acid), until the background stain was cleared.
6.3.6 LIGNIN ANALYSIS AND CHARACTERISATION

6.3.6.1 Lignin recovery
In order to recover the lignin fractions, fermentation broth (section 6.4.1.1) from the
following time points: 0, 24, 48 and 144 hours was vacum-filtered using a paper filter
(Whatman No. 1). Lignin was further dried and used for analysis.
287
6.3.6.2 Molecular weight (SEC)
Molar mass distribution determined using SEC was carried as described in section 3.3.2.6.
6.3.6.3 31P- NMR

31P- NMR analysis was carried out as described in section 3.3.2.3.
6.4 RESULTS
6.4.1 CHARACTERISATION OF LIGNINOLYTIC CAPABILITY OF P. SACCHAROPHILA IN THE PRESENCE OF CIMV
ORGANOSOLV LIGNIN
Based on results presented in section 5.4.2, P. saccharophila was selected from a range
of bacterial species as the strain exhibiting the highest ligninolytic capacity. In order to
further investigate its ability to produce ligninolytic enzymes, this strain was grown in
lignin supplemented minimal media and extracellular and intracellular proteins were
extracted from fermentation broth, quantified and analysed for ligninolytic activity.
6.4.1.1 P. saccharophila growth in M9 liquid media supplemented with CIMV lignin

P. saccharophila was inoculated into M9 liquid media supplemented with 0.25 % (w/v)
of CIMV lignin as the sole carbon and energy source and incubated at pH 7 and 30 oC for
144 hours (as described in section 6.3.3.1). The sampling times were based on the
incubation stages showing the highest concentration of phenolic compounds in previous
studies: 24, 48, 72, 96 and 144 hours (section 5.4.2). In order to prove the reproducibility
of the experimental design, cell growth was monitored via measurement of OD590 and
the growth curve was compared to the growth curve reported in section 5.4.1.
P. saccharophila grew rapidly in the first 24 hours, showed a slower growth rate between
24 and 72 hours and reached stationary phase between 72-144 hours of incubation. The
growth pattern was very similar to that reported in Chapter 5 (old curve) (Figure 6.2).
Liquid culture samples taken at different time intervals were extracted for protein.
288
0.7
Optical density @ 590 nM
0.6
0.5 PS new
0.4
PS old
0.3
0.2
0.1
0
0 50 100 150 200
Time (hours)
Figure 4.232: Growth of P. saccharophila in M9 liquid media supplemented with: 0.25 % (w/v) of CIMV
lignin for 144 hours at pH 7 and incubated at 30 oC.. PS new (growth curve for P. saccharophila from
study described in this section), PS old (growth curve for P. saccharophila grown in the same conditions
presented in section 5.4.1). Error bars represent standard deviation (SD±) of the mean (n=3).
6.4.1.2 Protein extraction and quantification

Extracellular and intracellular proteins were extracted from fermentation broth at the
following time intervals: 24, 48, 72, 96, 144 hours (as described in sections 6.3.5.1-
6.3.5.3). Concentration of protein was assayed using the Bradford method (as described
in section 6.3.5.4) and the results are shown in Table 6.2. The results of the assay showed,
that both extracellular and intracellular proteins were produced. However, the amount
of extracellular proteins for the first 72 hours of incubation was very low. The highest
concentration reaching 0.056 mg/mL was observed at 96 hours. After that, the amount
of proteins produced outside the cell markedly decreased. In turn, the concentration of
intracellular protein produced increased steadily over a time course of 144 hours.
Protein concentration reached a maximum amount of 1.816 mg/mL after 144 hours.
289
Table 4.162: Average concentration of proteins extracted at different time points with standard deviation
(SD±) of the mean (n=2).
I
Extracellular protein ntracellular protein concentration
Time concentration (mg/mL) (mg/mL)
24 hours 0.000 ± 0.000 1.307 ± 0.017

48 hours 0.005 ± 0.002 1.341 ± 0.017
72 hours 0.012 ± 0.002 1.494 ± 0.034
96 hours 0.056 ± 0.002 1.630 ± 0.034
144 hours 0.002 ± 0.002 1.816 ± 0.017
6.4.1.3 Protein expression

SDS-PAGE analysis of fractions collected over a time course of 144 hours, was carried out
as described in section 6.3.5.5. The molecular weights were determined based on pre-
stained standards run on a 4-20 % gradient gel (Bio-Rad, Hercules, CA, USA). Coomassie
blue staining of SDS-PAGE with crude extract showed multiple bands in the region
between 35-70 kDa, which corresponds with the reported size range for laccases and
dyp peroxidases. The protein bands in general were most intense at 96 hours of
incubation indicating maximum live cells at this point. Although proteins were not
detectable in most extracellular samples, several low intensity bands were observed in
fraction collected at 96 hours (Figure 6.3).
290
Figure 4.243: Coomassie Brilliant Blue-R-250 stained SDS-PAGE gel showing extracellular (A) and
intracellular (B) protein extracts recovered at different time points: 24, 48, 72, 96 and 144 hours. Red
squares include bands in the predicted laccase and dyp-peroxidsase size-range.
6.4.1.4 Assay for laccase activity

Extracellular and intracellular enzyme extracts of P. saccharophila grown in M9 liquid
media supplemented with 0.25 % (w/v) of CIMV lignin were screened for laccase activity
with ABTS. The activity was assessed visually by appearance of green colour in the assay
mixture and spectophotometrically by monitoring the increase in absorbance at 420 nm
(ε = 3.6 × 104 M–1 cm–1)(as described in section 6.3.4.1). Based on visual assessment and
spectophophotometrical analysis , laccase activity was not detected in these samples at
pH 5 and pH 7 (Table 6.3).
291
Table 4.173: Laccase activity of extracellular and intracellular enzymes extracted of P. saccharophila
grown in M9 liquid media with 0.25 % (w/v) of CIMV lignin determined visually and
spectophotometrically after 14 hours incubation at pH 5, pH 7 and 30 oC. Control -: no enzyme, Control
+: laccase from T. versicolor.
Type of
Laccase activity
enzyme
Time (hours)
pH 5 pH 7
24 48 72 96 144 C- C+ 24 48 72 96 144 C- C+
Extracellular
ND ND ND ND ND - + ND ND ND ND ND - +
- + ND ND ND ND ND - +
Intracellular ND ND ND ND ND
ND = not detected, + = positive for laccase activity, - = negative for laccase activity
6.4.2 THE EFFECT OF COPPER ON LACCASE ACTIVITY BY P. SACCHAROPHILA
Results from assay presented in section 6.4.1.4 showed, that laccase activity could not
be detected in extracts from P. saccharophila cultures. In order to evaluate whether
adding copper could enhance ligninolytic enzyme production and laccase activity by P.
saccharophila, the strain was grown in M9 media supplemented with 0.25 % (w/v) of
CIMV lignin and 0.25mM of CuSO4 for 144 hours (as described in section 6.3.3.1). A
control culture with no copper was also included. This growth experiment was carried
out using only one biological replicate. Following incubation, liquid cultures were
centrifuged to give cultures pellets and supernatants and both fractions were extracted
for protein (as described in sections 6.3.5.1-6.3.5.3). Three samples were taken at: 48, 72,
292
144 hours which were chosen for practical reasons, based on protein quantification
results presented in section 6.4.1.2.
6.4.2.1 Assay for laccase activity

Extracellular and intracellular enzymes produced by P. saccharophila in M9 liquid media
supplemented with 0.25 % (w/v) of CIMV lignin (control) and 0.25 % (w/v) of CIMV lignin
and 0.25 mM of CuSO4 (test) were screened for laccase activity (as described in section
6.3.4.1). As observed above, laccase activity was not detected by either visual
assessment or spectophophotometrical analysis at pH 5 or pH 7. In order to exclude a
possible inhibitory effect of McIlvaine buffer on enzyme activity, the screen was
repeated with 0.1 mM phosphate buffer at pH 6. In these conditions, rapid development
of a green colour was observed with enzyme extracts, indicating positive laccase activity.
Microtitre plates were incubated for 14 hours for maximum colour development. With
respect to enzymes extracted from media supplemented with CuSO4, the highest activity
was observed with intracellular enzymes extracted after 72 hours of incubation.
Intracellular enzymes extracted after 144 hours of incubation showed moderate activity.
Laccase activity was not detected in the 48 hour sample. Extracellular enzymes extracted
after 48 and 72 hours of incubation exhibited clear but very low activity. Laccase activity
was not detected in the 144 hour samples. In comparison to enzymes extracted from
control cultures, inclusion of CuSO4 resulted in higher laccase activity in intracellular
samples recovered after 144 hours of incubation. This treatment also showed earlier
expression of extracellullar laccase activity which was detected after 48 hours of
incubation (Table 6.4). Due to the formation of precipitate, it was not possible to obtain
accurate spectrophotometric readings.
293
Table 4.184: Laccase activity of extracellular and intracellular enzymes produced by P .saccharophila in
M9 liquid media with 0.25 % (w/v) of CIMV lignin determined visually at pH 6 and 30 oC in control and
test experiment. Control: - no enzyme, Control +: laccase from T. versicolor.
Type of enzyme Laccase activity

(
Control Test
experiment experiment
(-copper) (+copper)
Time (hours)
48 72 144 C+ C- 48 72 144 C+ C-
Extracellular ND + ND P N + + ND P N
Intracellular
ND +++ + P N ND +++ ++ P N
ND = not detected, P = positive for laccase activity, N = negative for laccase activity, + = low activity, ++ = moderate activity,
+++ = high activity
6.4.3 CHARACTERISATION OF CIMV LIGNIN MODIFIED BY P. SACCHAROPHILA
Results from sections 6.4.1 and 6.4.2 showed, that P. saccharophila has the ability to
utilise CIMV lignin as the sole carbon and energy source and produce ligninolytic
enzymes, including laccase. In order to evaluate modifications in lignin structure
mediated by the P. saccharophila strain, lignin was recovered from culture broth (section
6.4.1.1) (as described in section 6.3.6.1) at different time intervals and subjected to basic
analysis. Due to time constraints, analysis was carried out on only one lignin sample
recovered from the 4 time points: 0, 24, 48 and 144 hours (these times were chosen for
practical reasons). Molecular weight of lignin fractions was measured by Size Exclusion
Chromatography (SEC) (as described in section 6.3.6.2). Analysis of functional groups was
carried out by Phosphorus-31 NMR spectroscopy (31P- NMR) (as described in section
6.3.6.3).
294
6.4.3.1 Molecular weight distribution (SEC)
The molecular weight distribution of lignin samples was obtained by Size Exclusion
average (Mn), Weight- average (Mw) molecular weights and polydispersity (Mw/Mn) of
the lignin samples recovered after 0, 24, 48 and 144 hours of incubation with P.
saccharophila are shown in Table 6.5. As can be seen below, the Mm, Mw and Mw/Mn
ranged from 1213-1258 Dalton, 7585-7988 Dalton and 6.3-6.4, respectively. The
molecular weights steadily increased with the incubation time. The highest values of
average weight molecular masses were observed for CIMV lignin recovered after 144
hours of incubation (Mn: 1258 Dalton, Mw: 7988 Dalton) and the lowest for CIMV lignin
recovered at 0 hours (Mn: 1213, Mw: 7585). All lignin fractions showed a comparable
degree of polydispersity (Mw/Mn: 6.3-6.4), which was higher compared to Alcell lignin
(Mw/Mn:4.3) and lower compared to CIMV wheat straw lignin (Mw/Mn: 9.5).
Table 4.195: Average molar mass data (Mw, Mn) and polydispersity (Mw/Mn) of lignin samples modified
by P. saccharophila in M9 liquid media. Standard deviation (SD) of the mean (n=2) is 0.
CIMV lignin 0HR 1213 7585 6.3

CIMV lignin 24 HR 1232 7907 6.4
CIMV lignin 48 HR 1258 7955 6.3
CIMV lignin 144 HR 1258 7988 6.3
CIMV lignin 907 8602 9.5
Alcell 681 2948 4.3
6.4.3.2 31P- NMR

6.3.6.3. Table 6.6 shows the amount of hydroxyl groups as determined by 31P-NMR,
consisting of aliphatic OH, condensed phenolic OH, syringyl OH, guaiacyl OH, p-hydroxyl
OH, and COOH groups. The concentration of each hydroxyl functional group (mmol g-1)
was calculated on the basis of the hydroxyl content of the internal standard and its peak
area). CIMV lignin at 0 hours had the highest amount of total OH groups according to
31P-NMR data (3.67 mmol g-1). The lowest content of total OH groups in the lignin
295
samples (3.04 mmol g-1) was observed in the case of CIMV lignin recovered after 24 hours
incubation (Figure 6.4). Aliphatic OH groups formed the largest component of total OH
content with the highest content observed in lignin recovered at 144 hours incubation
(1.27 mmol g-1) (Figure 6.5). The total content of hydroxyl groups markedly decreased
between 0-24 hours of fermentation, and increased again between 48-144 hours. There
were no notable differences in the amount of functional groups between 24-48 hours of
incubation. With respect to guaiacyl OH groups and COOH groups, the highest amount
was observed in lignin sample recovered at 0 hours. In turn, the highest amount of
aliphatic OH groups, condensed phenolic OH groups and syringyl OH groups was
observed in lignin samples recovered after 144 hours of incubation. Lignin standards
composition varied with that of culture samples showing different distrubutions of OH
groups. The highest total OH content was observed for Kraft Indulin AT lignin (6.30 mmol
g-1) which had the highest content of aliphatic, condensed phenoic and guaiacyl OH
groups compared to other fractions. P1000 (Protobind) lignin showed the highest
content of COOH groups (1.05 mmol g-1) and Alcell had the highest content of syringyl
OH groups (1.05 mmol g-1) (Table 6.6).
296
Table 4.206: Contents of functional groups as determined by 31P- NMR. The range of OH groups include: aliphatic OH, cond.Phen.OH, syringyl OH, guaiacyl OH, p-Hydroxyl OH
and COOH (mmol g-1). Standard deviation (SD) of the mean (n=2) is 0. ND= not detected.
Aliphatic Cond. Phen

Time (hours) Syringyl OH Guaiacyl OH p-Hydroxyl OH COOH Total
OH OH
0 1.26 0.53 0.30 0.58 0.31 0.69 3.67
1.13 0.42 0.28 0.52 0.24 0.44 3.04

24
1.15 0.44 0.28 0.52 0.24 0.44 3.05

48
1.27 0.54 0.31 0.57 0.31 0.55 3.54
144
Alcell 1.08 0.76 1.05 0.70 0.20 0.30 4.09

P1000(Protobind) 1.73 0.70 0.56 0.70 0.45 1.05 5.19
Kraft Indulin AT(Softwood) 2.35 1.36 N/D 1.88 0.22 0.49 6.30
297
Figure 4.254: Total contents of functional groups as determined by 31 P-NMR in mmol g-1. Standard
deviation (SD) of the mean (n=2) is 0.
Functional group content

1.4 Aliphatic OH
1.2
1 Cond. Phen OH
0.8
Syringyl OH
0.6
0.4 Guaiacyl OH
0.2
0 p-Hydroxyl OH
CIMV lignin 0HR CIMV lignin 24 HR CIMV lignin 48 HR CIMV lignin 144
HR
COOH
Lignin
Figure 4.265: Contents of functional groups in different lignin fractions. Data represent aliphatic OH,
cond.Phen.OH, syringyl OH, guaiacyl OH, p-Hydroxyl OH and COOH groups in mmol g -1. Standard
deviation (SD) of the mean (n=2) is 0.
298
6.5 DISCUSSION
Bacteria are a rich source of potential industrial enzymes and due to their rapid growth,
reasonable production costs, extent of enzyme complexity and biodiversity offer
advantages over alternative sources (Fisher and Fong, 2014). Although a number of
studies have already shown, that bacteria can directly degrade lignin, studies on enzyme
pathways are limited (Ahmad, 2010). Hence, there is a large focus on the determination
of the enzymatic basis of bacteria-mediated lignin conversion in industrial biotechnology
(Ghribi et al., 2016).
In this study, P. saccharophila was examined in detail for the ability to utilize CIMV lignin
and produce ligninolytic enzymes. The gram-negative bacteria, commonly known as
Pseudomonas saccharophila, recently reclassified as Pelomonas saccharophila was
chosen from a range of bacterial species which were investigated for their capacity for
bioconversion of CIMV organosolv presented in Chapters 4 and 5. Results obtained here
confirmed the robustness of the experimental design with cell growth, monitored via
measurement of OD590, showing good agreement with the results presented in section
5.4.1 (Figure 6.2).
Protein extracts from fermentation broth taken at the following time points: 24, 48, 72,
96, 144 hours were quanitified for protein according to the method of Bradford (1976).
The Bradford assay, also known as Coomassie brilliant blue protein assay, enables rapid
and simple total protein quantification in a sample. The results of the assay carried out
in this study showed, that intracellular proteins in P. saccharophila cultures increased
steadily over the time course reaching a maximum of 1.816 mg/mL after 144 hours. In
contrast, extracellular proteins showed a different pattern of accumulation with very low
levels detected in the first 72 hours of incubation and the highest concentration
observed after 96 hours of incubation (0.056 mg/mL) and marked decline by 144 hours.
It is of interest, that the maximum levels of intracellular proteins at 144 hours
corresponded with a low extracellular protein content (Table 6.2). P. saccharophila
exhibited maximum growth rate during first 72 hours of incubation and reached the
stationary phase around 72-144 hours (Figure 6.2). This could potentially indicate, that
extracellular enzymes were only produced during the active growth period. After that, it
299
appears that extracellular proteins were either degraded or absorbed by the bacteria
during the stationary phase. Ohta et al., (2015) proposed, that extracellular enzymes are
typically involved in the primary stages of bacterial lignin modification. Formation of
extracellular enzymes in the exponential phase was demonstrated with B. subtilis (Liebs
et al., 1988). In turn, according to Reed, (1966), intracellular enzymes are known to be
produced during logarithmic phase, but released to media during the declining phase, as
cells start to undergo lysis. Lack of an observable increase in extracellular proteins during
the stationary phase indicates there was limited lysis of P. saccharophila cells in these
experiments.
SDS-PAGE is an electrophoretic technique widely used in biotechnology, biochemistry,

molecular biology and other life science laboratories. Using this technique, proteins are
separated on a polyacrylamide gel based on their molecular weight. Hence, this analysis
can be used to characterize protein molecules in terms of their molecular weight and
monomeric or dimeric forms (Saraswathy, 2011). SDS-PAGE analysis of fractions
collected over a time course of 144 hours showed changes in protein profiles with time.
Intracellular proteins exhibited the highest intensity bands at 96 hours and extracellular
protein also gave rise to faint bands for the same time point (Figure 6.3). The above
findings corroborated results for protein concentration measurements (section 6.4.1.2)
showing the highest intensities of intracellular and extracellular proteins between 96-
144 and at 96 hours, respectively (Table 6.2). It is worth mentioning, that protein profiles
showed multiple bands in the region between 35-70 kDa which covers the size-range
reported for laccases and Dyp-type peroxidases (Kuddus et al., 2013; Arunkumar et al.,
2014; Li et al., 2012). Laccases exhibiting molecular masses in a range of 35-50 kDa have
previously been purified from Pseudomonas species. According to Kuddus et al., (2013),
a laccase from P. putida migrating as a single band in SDS-PAGE showed a molecular mass
of 39.5 kD. In turn, Arunkumar et al., (2014) purified a laccase from P. aeruginosa which
gave rise to a single band corresponding to 43 kDa. The Dye-type peroxidase from
P.aeruginosa exhibited a molecular mass of 64kDA (Li et al., 2012).
Extracellular and intracellular enzymes were analysed for laccase activity. Although
bacterial laccases are less well studied than those in fungi, they are still widespread.
Recent studies revealed over 100 different bacterial laccase sequences identified from
300
DNA that had been isolated from forest soil (Kellner et al., 2008). Studies carried out by
Alexandre and Zhulin, (2000) revealed, that laccase might be one of the key ligninolytic
enzymes produced by Pseudomonas sp. Based on visual assessment and
spectrophotometric analysis, no laccase activity was detected in samples assayed at pH
5 and pH 7 with McIlvaine buffer (Table 6.3). Laccase production is mostly influenced by
physical and chemical environment of cultural conditions, such as quality and quantity
of carbon and nitrogen source, pH, temperature, aeration, presence of inducers,
vitamins, amino acids, phosphorus, metal ion etc (Nandal et al., 2013). Hence, for
effective laccase production, it is essential to simultaneously optimize the culture
conditions and composition of media.
In order to evaluate whether adding copper could enhance ligninolytic enzymes

production and laccase activity by P. saccharophila, the test strain was grown in M9
media supplemented with 0.25 % (w/v) of CIMV lignin and 0.25mM of CuSO4 for 144
hours. Copper is an essential micronutrient for most living organisms and required for
assembling copper proteins, which are involved in oxidation and reduction reactions. It
is important to note, that laccases are metallo-enzymes containing four copper atoms
per molecule. Additionally, copper acts both as an inducer and micronutrient, hence it
has a potential to increase laccase production considerably (Rajeswari and
Bhuvaneswari, 2016). Recent studies carried out by Nakade et al., (2013) concluded, that
fungal laccase activity in the presence of CuSO4 was 20 times higher than in the absence
of this inducer. In current study, CuSO4 was added to the media at the start of the
incubation. It has been suggested, that addition of an inducer during the exponential
growth phase rather than stationary phase can achieve the maximal stimulatory effect.
A control culture with no inducer was also included.
Extracellular and intracellular enzymes produced by P. saccharophila in both samples

were screened again for laccase activity. The results of the assay carried out at pH 5 and
pH 7 with McIlvaine buffer (section 6.4.2.1) corroborated the results of the previous
experiment (6.4.1.4). This could potentially indicate an inhibitory effect of citric acid
present in McIlvaine buffer. According to Lorenzo et al. (2005) different organic
compounds present in the environment can affect the stability of ligninolytic enzymes.
Organic compounds such as citric acid, oxalic acid, malonic acids are copper-chelating
301
agents and can modify laccase active sites by forming complex compounds with its
copper ions. In order to exclude the potential inhibitory effect of citric acid on the
enzyme expression, the assay was repeated in 0.1 mM phosphate buffer at pH 6. In
studies presented by Chefetz et al. (1998), phosphate buffer pH 6 has previously shown
enhancement of laccase activity purified from Chaetomium thermophilium. In this
research, the results of the assay performed in phosphate buffer showed rapid
development of green colour, indicating laccase activity in enzymes extracted from both
control and copper supplemented media. Activity was highest with intracellular protein
extracts (Table 6.4). Rajeswari and Bhuvaneswari (2016) showed previously that
extracellular bacterial laccases are constitutively produced in small amounts.
Although the enzyme activity was expressed in control cultures, addition of CuSO4 to the
growth medium markedly increased intracellular laccase production after 144 hours of
incubation. Additionally, the presence of CuSO4 resulted in the production of
extracellular laccase production by 48 hours incubation (Table 6.4). Compared to fungal
laccase induction, publications on bacterial induction through addition of copper
supplements are relatively scarce (Asina et al., 2017). According to Rajeswari and
Bhuvaneswari (2016), the amount of copper required to induce laccase activity varies
with different bacterial species, ranging from 0.1mM to 1 mM. However, Zheng et al.,
(2013) recently showed, that supplementing media with 3 mM CuSO4 induced laccase
production by Proteus hauseri. Additionally, Rajeswari and Bhuvaneswari, (2016) also Field Code Changed
showed, that 1 mM CuSO4 increased laccase production by Bacillus sp.
The presence of laccase activity in phosphate buffer confirmed the theory about the
inhibitory effect of citric acid. Reiss et al., (2011) previously showed that incubation of
laccase produced by Bacillus pumilus in McIlvaine buffer at pH 5 and pH 7 for 1 hour
showed respectively 75 % and 50 % loss of activity. In turn, studies carried out by Yuan
et al. (2016) revealed that almost no activity was observed when incubating laccase in
McIlvaine buffer pH 4 for 6 hours.
In order to fully confirm the ligninolytic capability of P. saccharophila, CIMV lignin

samples were recovered from fermentation broth at different time intervals and
subjected to basic analysis. Tolbert et al., (2016) previously postulated, that bacteria are
suitable tools for modification of the molecular weight of lignin. The results of SEC
302
analysis presented here showed a slight increase in the molecular weight with incubation
time (Table 6.5). There are several reasons which could explain this outcome. The initial
increase of Mw and Mn could be due to lignin polymerization. According to Abdelaziz et
al., (2016) when laccase is involved in lignin bioconversion it oxidises lignin, generating
aromatic radicals that can undergo further reactions and eventually repolymerize lignin.
Similar finding have been presented in the literature. The formation of high molecular
weight lignin treated with laccase was recently reported by Edalatmanesh et al., 2012. It Field Code Changed
is worth mentioning, that the average molecular mass of CIMV lignin recovered at 0
hours was higher than the CIMV lignin standard. This was most likely a result of thermal
mediated changes during the sterilisation process. Taking into account that the
incubation of bacteria was initiated with partially depolymerised lignin it is also possible,
that production of low-molecular weight compounds was increased and their rapid
consumption left high molecular weight lignin in the fermentation system (Salvachúa et
al., 2015). Alternatively, lower molecular weight break-down products may have
remained into solution and not been recovered in the lignin fraction. Metabolism of
products derived from lignin presented in section 5.4.2, could potentially support this
theory. It is of interest, that mass spectrometry analysis of lignin break-down products
presented in section 5.4.2 focused on molecules with a molecular weight <500 and SEC
analysis was carried out with solid lignin fractions. Therefore, soluble oligomeric lignin
may have been missed out during analysis. This would be an important area to focus on
in future studies to fully explain P. saccharophila metabolism of CIMV lignin.
The results of 31P-NMR analysis indicated, that ligninolytic enzyme activity induced
modification of lignin structure. It is of interest, that the amount of all classes of hydroxyl
groups decreased up to 48 hours incubation and increased again between 48-144 hours
(Table 6.6). These outcomes correspond with growth patterns observed for P.
saccharophila cultures which showed a stationary phase between 24-48 hours followed
by an increase in growth up to 72 hours (Figure 6.2). The decrease in COOH groups
during incubation indicated that changes were not related to oxidative activity as this
would cause an increase in this class. It is well documented, that carboxylic groups
increase as a consequence of oxidative enzyme activity It must be noted that cleavage
of ferulic and coumaric acid would result in an decrease in in lignin COOH content
303
however this would also cause a drop in average molecular weight. In turn, an increase
in COOH groups between 48 and 144 hours of incubation could suggest oxidative activity
however this should result in a decrease in aliphatic hydroxyl groups which was not
observed. Moreover, it is predicted that this would induce repolymerisation which would
cause a reduction in other hydroxyl groups and increased polydispersity which did not
occur. Cleavage of linkages would also cause an increase in functional groups however,
the average lignin molecular weight increased between 48 and 144 hours. It is likely, that
a combination of factors account for these changes however differences were small and
may not have been significant. If bacterial metabolism is mainly occurring with a soluble
oligomeric lignin pool as proposed above, it would be of interest to investigate changes
in functional groups in this fraction.
This study provided a further insight into the enzymes involved in P. saccharophila-
mediated conversion of CIMV lignin. Elucidation of the bacterial enzyme systems for
lignin degradation is important not only in terms of the Earth’s carbon cycle but also for
providing useful tools for the conversion of lignin into products of industrial value (Masai
et al., 2007). Industrial enzymes are currently at the heart of green chemistry as
illustrated by a global market value that reached 5 G$ in 2013 and is expected to have
grown to 7 G$ by 2018 (Ghribi et al., 2016).Laccase, one of the most abundant ligninolytic
enzymes has already received much attention due to its ability to oxidise a broad
spectrum of organic compounds (de Gonzalo et al., 2016). Apart from this, laccase also
demonstrated the ability to catalyse reactions where various organic compounds were
grafted onto lignin derivatives which sequentially led to modified properties of the lignin
derivatives (Gillgren et al., 2017). Additionally, Mai et al., (2000) recently suggested, that
laccase can be used to synthesize a co-polymer of organosolv lignin (LO) and synthetic
monomer acrylamide to produce new plastic-like engineering materials. Therefore, the
optimum conditions for the laccase production should still be investigated for its cost
effective commercial production (Nandal et al., 2013).
304
6.6 CONCLUSIONS
This study investigated the enzymatic basis of P. saccharophila-mediated organolov

lignin conversion. The results of the analysis revealed, that this species has the ability to
produce ligninolytic enzymes to modify and degrade lignin. For the first time,
intracellular and extracellular laccase activity with P. saccharophila was identified.
Enzyme activity was detected with phosphate buffer pH 6. It is postulated, that the lack
of enzyme expression in McIlvaine buffer could be caused by the inhibitory effect of citric
acid. Although the presence of CuSO4 was not essential, its inclusion induced more rapid
expression of extracellular laccase and increased intracellular laccase production
towards the end of incubation. The physical and chemical modifications in lignin
structure revealed by SEC and 31P-NMR analysis indicated changes in the molecular
weight distribution of this pool. Overall results confirmed, that P. saccharohila is a
potential candidate for bioconversion of CIMV organosolv lignin and further use in
biorefining. However, follow up studies should be performed to identify unexplored
enzymes produced by this species, to fully characterise changes in lignin fractions and
develop processes for lignin conversion to high-value products.
305
Chapter 7
306
Chapter 7: GENERAL DISCUSSION
7.1 AIMS AND BACKGROUND
Lignocellulosic biorefineries are currently considered as the best alternative to promote

the transition from a fossil fuel-based economy to a bio-based one for a sustainable
future. However, the commercialization of a biomass-based biorefinery is largely
dependent on the exploitation of all biomass components. The cellulose and
hemicellulose fractions, which are both a source of sugars, are commonly used in the
production of biofuels and synthesis of surfactants or polymers (Gosselink, 2011). Lignin,
as a highly complex phenolic polymer, has been traditionally viewed as a 'waste product'
in paper and biofuel production (Lange et al., 2013). However, the chemical structure of
lignin indicates that it might be a potential source of high-value products (Cotana et al.,
2014). Therefore, identification of suitable methodology to convert lignin into industrial
materials and value added aromatic platform chemicals is an important target to
improve the economic profitability of biorefining (Ayixiamuguli, 2016).
The main aim of the study presented in this thesis was to identify a comprehensive range
of processing technologies for the generation of industrially useful forms of lignin with
the aim of encouraging commercialization of lignin and helping to advance this area of
research.
First attempts were focused on extraction of lignin fractions from different feedstocks
and processes focusing on high extraction yield and analysis of generated fractions in
terms of suitability for the production of biomaterials. For the purpose of the
experimental research programme, lignin was sourced directly from feedstocks utilized
in the ADMIT Bio-SuccInnovate work packages and from fibre fractions generated in
biorefining processes developed at the Biorefining Centre of Excellence. In order to carry
out full valorization, lignin fractions were extracted from grasses and wood (Chapter 2).
Comprehensive analysis of lignin composition and functionalities carried out by wet
chemical analysis, FT-IR, TGA, 31P-NMR and SEC (Chapter 3) revealed, that some fractions
have a large potential for production of polyurethanes, phenolic resins and polyesters.
307
Another aspect of this project was to test the potential for microbial bioconversion of
lignin into valuable chemicals and building blocks for bio-based products. Novel
screening methods were developed in order to identify fungi and bacteria capable of
metabolising technical lignin (Chapter 4). An insight into the mechanism of microbial-
mediated lignin degradation was also provided to understand the biochemistry and
structure-function relationships of different pathways involved in this process (Chapter
5). In the end, the ligninolytic capability of the most ‘active’ strain towards lignin
degradation was examined in detail in a series of laboratory-scale fermentation
experiments (Chapter 6). This part of the research pointed the feasibility of using various
microorganisms to convert lignin into high-value chemicals used in food and chemical
industry and building blocks for the production of biomaterials.
7.2 KEY FINDINGS
 Both organosolv and a combined steam explosion-organosolv process exhibit

high lignin extraction efficiency for grasses and hardwood yielding 67.23 % -
93.27 % of total lignin content.
 Combined steam explosion-organosolv process is the most suitable for lignin
extraction from Miscanthus giganteus.
 Steam explosion with acid treatment increases the extraction efficiency yield
with most of the feedstocks.
 Steam explosion with alkaline treatment does not increase extraction efficiency
yield with most of the feedstocks investigated here.
 Organosolv process and combined steam explosion-organosolv process is not
suitable for lignin extraction from pine.
 Lignin composition depends on feedstock and treatment.
 MG SE ALk lignin, WS SE Alk lignin, WS lignin, MG lignin, PN lignin, PN SE Acid
lignin reveal a high content of total OH groups, aliphatic groups and high solubility
in dioxane and DMSO indicating good potential for use in polyurethanes.
 WS SE Acid lignin, WS lignin, CS lignin, PN SE Alk lignin, PN SE Acid lignin, WW SE
ALk lignin reveal low-molecular weight and low polydispersity, indicating good
potential for incorporation into phenol-formaldehyde resins (PF resins).
308
 The broad range of desirable properties observed for WS SE Acid lignin and PN
SE Acid lignin shows that they have potential as a source of raw material for
production of polymers such as polyurethanes, polyesters and PF resins.
 T. harzianum, P. putida, P. saccharophila and P. fluorescens have the ability to
utilize CIMV lignin as the sole carbon and energy source and produce ligninolytic
enzymes.
 The fungal and bacterial strains investigated here use fundamentally different
strategies for lignin utilization.
 T. harzianum, P. putida, P. saccharophila and P. fluorescens metabolism of CIMV
lignin gives rise to break-down products and other lignin-related phenolics
indicating cleavage of ester and β-ether linkages and catabolism of
phenylcoumarans.
 P. saccharophila produces extracellular and intracellular laccase.
 Addition of CuSO4 induces earlier expression of extracellular laccase and
increases intracellular laccase production in the latter stages of incubation in P.
saccharophila cultures.
 Out of 4 different species investigated here, P. saccharophila shows the greatest
potential for bioconversion of CIMV lignin and production of high-value
chemicals and building blocks for biomaterials.
7.3 GENERAL REMARKS, FUTURE PERSPECTIVES AND RESEARCH DIRECTIONS
There is currently a major focus on the development and implementation of biorefinery

processes to achieve a sustainable economy based on bioresources (Menon and Rao,
2012). In this research, new processes based on organosolv technology were developed
to add value to lignin, a by-product generated by one of the project partners. These Commented [AW[148]:
Commented [AW[149R148]: Maybe safer to not to specify
processes enabled production of new lignin fractions from grass and wood focusing on steam explosion as you look at both with and without
high extraction yield and optimal composition for production of industrial materials. At
present, there are no reports in the literature detailing the effect of steam explosion on
organosolv lignin extraction from Miscanthus, wheat straw, corn stover, and willow.
Therefore, this is the first study revealing the potential of a two stage process for lignin
309
extraction as an integral part of a biorefinery concept. In contrast, the steam explosion-
organosolv procedure proved ineffective for lignin extraction from pine. Hence, further
method development is required to optimise extraction from this feedstock and options
include addition of extra catalysts, optimization of temperature, time and the
appropriate ratio of solvents mixture.
Lignin-derived products can potentially be used for multiple applications and the
chemical and physical properties of lignin extracts determine their suitability for different
applications. Thus there is a need for detailed analysis of lignin fractions prior to
application development (Gosselink, 2011). Physico-chemical analysis of WS SE Acid and
PN SE Acid lignin fractions revealed their potential for the production of biomaterials
such as polyurethane and phenol-formaldehyde resins (PF resins). Therefore, future
research should ideally involve small-scale trials incorporating generated lignin fractions
into these types of bio-products. Currently, new sulphur-free lignin products such as
organosolv lignin are preferred overall to commercially available lignin fractions (Kraft
and lignosulphonate) (Tuomi et al., 2010). Therefore, successful replacement of fossil-
based chemicals with lignin would certainly have an impact on future development of
lignocellulosic biorefineries. Moreover, it would also provide economic incentives by
creating green jobs and boost competition on the energy market by offering energy
alternatives.
Enzymatic modification and degradation of lignin is considered a potential target for

process development because of the mild nature and substrate specificity of the
enzymes as well as the ability to preserve the integral structure of lignin compared with
chemical oxidation (Ayixiamuguli, 2016). In this study, development of new screening
methods enabled identification of microorganisms able to utilise CIMV lignin as the sole
carbon and energy source, revealing their promising potential in industrial
biotechnology. However it is considered, that the growth conditions for P. putida,
P.saccharophila, P. fluorescens as well as T. harzianum should be further optimized and
that screening methods should be expanded in order to identify a larger variety of lignin-
degrading enzymes and to characterise their activity. The options include using different Commented [AW[150]: Options include diff buffer systems,
pH, more concentrated enzyme extracts
buffer systems, pH and more concentrated enzyme extracts. According to Asina et al.,
(2017) enhanced bioconversion of lignin could also be achieved by co-culturing of
310
different microorganisms. Their hypothesis is that the competition for limited space and
nutrients, may result in an improved degradation performance through elevated
ligninolytic enzyme production. The future objective is that optimisation of the
bioconversion process will improve the physico-chemical properties of lignin fractions
generated using methods described here and increase their suitability for incorporation
into biopolymers.
Analysis of low molecular weight lignin break-down products proved a useful tool for
providing deeper insights into microbial-mediated CIMV organosolv lignin metabolism.
This study showed, that for the microbial strains investigated here, the main pathways
of lignin metabolism involved esterase and β-etherase activity and the phenylcoumaran
catabolic pathway. Of the four studies species, P. saccharophila showed the best
potential for future use in biorefining. This species produced relatively high amounts of
break-down products combined with higher growth rates indicating effective
metabolism of the lignin substrate. The products such as ferulic acid and GHP have
potential for the synthesis of high-value chemicals used in the food and chemical industry
and building blocks for the production of polyesters, polycarbonates, epoxy and
urethane resins. It must be noted that results of analysis of lignin structure modifications
mediated by the P. saccharophila strain were inconclusive. It is proposed, that a soluble
oligomeric lignin pool was metabolised in the bacterial culture and that this may have
escaped detection as mass spectrometry focused on molecules with Mw <500 and lignin
characterisation studies were carried out on the solid lignin fraction. Future studies
should focus on this lignin pool in order to gain a full understanding of metabolic
pathways involved in lignin utilisation by this strain.
An important outcome of this research is that it provides the first reported evidence for
the production of intracellular and extracellular laccase activity by the P. saccharophila
strain. Due to project timeline and funding constrains, further ligninolytic capability of P
saccharophila could not be explored. However, there are several experimental
approaches which could enrich the conclusions. First of all, further work would require
full understanding of laccase and characterization of lignin fractions modified with this
type of enzyme. Additionally, genome sequencing should be carried out in order to
reveal a range of other enzymes involved in lignin conversion and their impact of
311
production of bio-based products. Eventually, biosynthetic pathways could be
engineered so the valuable bio-products would accumulate to higher levels under a
wider range of fermentation conditions.
Overall, the findings of these studies indicated that P. saccharohila would be a promising
candidate in the modern biorefinery. It is proposed, that purified, lignin-degrading
enzymes from this strain could be commercially applied to the pre-treatment of
lignocellulosic biomass and more specifically lignin conversion which may yield an array
of building blocks and renewable aromatic chemicals for use in a range of applications in
green chemistry and biotechnology.
Outcomes of the research carried out during this PhD programme have generated
sufficient data for a range of per-reviewed publications. Currently, there is ongoing work
on mini-review manuscript intending to summarize recent advances and issues involving
the use of lignin as a building block for polymer materials. Additionally, two other
manuscripts focusing on the comparative studies of the effect of steam explosion on
lignin extraction from grass and wood feedstocks and their suitability for synthesis of
high-value products are being prepared. At a subsequent stage, the preparation of
another manuscript investigating the mechanism of microbial-mediated CIMV
organosolv break-down and ligninolytic capability of P. saccharophila is also planned.
Taking into account that there were no previous publications in this area of research,
there is a hope that it may lead to new perspectives on further applications of lignin.
Despite extensive interest in lignin valorisation around the world, using both physico-
chemical and biological approaches, there is currently no clear consensus about what
type of lignin preparation is optimum for subsequent lignin valorization (Lancefield et al.,
2016). In order to make lignin-based products technologically and economically viable,
specific objectives need to be achieved (Boeriu et al., 2014). Firstly, extensive process
development and optimization is required to enhance the efficiency of conversion of
lignin containing waste streams. Secondly, extensive screening of microorganisms and
engineering is needed to develop microbial processes for effective depolymerization and
conversion of lignin to various products. In the context of an integrated biorefinery based
on lignocellulosic feedstocks, effective pre-treatments need to be incorporated into the
overall process to enable maximum and efficient extraction of components from the
312
biomass. Ultimately, techno-economic and life cycle analysis is essential to thoroughly
evaluate the economic and sustainability of the process (Xie et al., 2016). There has been
large investment into research and development on the integration of lignin into bio-
based products over the last 10-15 years but processes developed at a research level
have not reached a meaningful industrial scale (Laurichesse and Avérous, 2013).
Research described here will contribute to these aims in terms of process development
for lignin extraction and screening of micro-organism for bioconversion of lignin and will
be useful for creating new and advanced technologies and lignin-based products.
313
7.4 CONCLUSIONS
Overall, attempts to identify a comprehensive range of technologies for the generation

of industrially useful forms of lignin have been successfully accomplished. The results
presented in this thesis have contributed to a better understanding of CIMV organosolv
lignin chemistry and structure, and process development for the extraction of lignin. In
addition, opportunities for microbial conversion of CIMV lignin into different types of
bio-products have been unveiled. Moreover, some lignin fractions produced from the
combined steam explosion-organosolv process showed properties tailored to
commercial production of phenolic resins, polyurethanes or polyesters based on lignin
macromolecule. It is worth noting, that research carried out by other project partners,
as part of the ADMIT Bio-SuccInovate project, has already demonstrated the feasibility
of converting other components of the lignocellulosic biomass into xylitol and succinic
acid. Research described here provides a route for valorisation of the lignin waste
streams generated by these processes. Hence, the outcome of this study can potentially
contribute to the development of the European bioeconomy. Outcomes of research
presented in this thesis, in combination with other research efforts by the global
community should ultimately lead to the commercial utilization of lignin on a large-scale.
It is only a matter of time before research and development in this area can be
implemented in practice, but the old joke ‘‘you can make anything out of lignin...except
of money‘‘ has already been proven wrong. It seems to be justified, that lignin will
become a major raw material for the chemical industry and a relevant source of profit
for the bioeconomy.
314
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APPENDIX
Figure 1: Growth of T. harzianum (mycelium) in YNB liquid media supplemented with different amounts of Miscanthus giganteus (MG) at pH 5 and 28 oC. Test: A) YNB+ 25 % (w/v) MG (milled),
B) YNB+ 25 % (w/v) MG (not milled), C) YNB + 10 % (w/v) MG (not milled), D) YNB + 5 % (w/v) MG (not milled), Control -: YNB and inoculum , Control + : YNB with 0.5 % (w/v) of glucose and
inoculum. Commented [AW[151]: What is difference between 1 & 2?
346
Figure 2: Growth of T. harzianum (spores) in YNB liquid media supplemented with different amounts of Miscanthus giganteus (MG) at pH 5 and 28 oC. Test: A) YNB+ 25 % (w/v) MG (milled), B)
YNB+ 25 % (w/v) MG, C) YNB + 10 % (w/v) MG, D) YNB + 5 % (w/v) MG, Control -: YNB and inoculum, Control +: YNB with 0.5 % (w/v) of glucose and inoculum.
347
Figure 3: Laccase activity of extracellular enzymes produced by T. harzianum after incubation in YNB liquid media
supplemented with 25 % (w/v) of Miscanthus giganteus at pH 5 and 28 oC. Test: Buffer + ABTS, enzyme extract, SDS,
Control -: Buffer + ABTS, SDS, Control +: Buffer, ABTS, laccase from T. versicolor.
348
Figure 4: Assay for ligninolytic enzyme production by T. harzianum on YNB solid media supplemented with methylene blue (0.05 g/l) at pH 5 (on the left) and pH 7 (on the right), after 5 days of
incubation at 28 oC . Test: inoculated plate, Control - : uninoculated plate.
pH 5 pH 7
349
Figure 5: Assay for ligninolytic enzyme production by T. harzianum on YNB solid media supplemented with different amounts of CIMV lignin: A) 0.25 % (w/v), B) 0.5 %(w/v), C) 1% (w/v) and
methylene blue (0.05 g/) at pH 5 (on the left) and pH 7 (on the right), after 5 days of incubation at 28 oC .Test: inoculated plate, Control - : uninoculated plate, Control +: plate supplemented with
laccase from T.versicolor.
pH 5 pH 7
350
Figure 6: Assay for ligninolytic activity (based on methylene blue decolourisation) in spent culture supernatants after incubation of T. harzianum in YNB with different amounts of CIMV lignin: A)
0.25 % (w/v), B) 0.5 % (w/v), C) 1 % (w/v), D) 2 % (w/v) for 10 days at pH 5 and 28 oC. Test: Buffer+ spent culture supernatant +methylene blue solution. Control –: (1) Buffer+ methylene blue
solution, (2): Buffer + YNB (no enzyme), methylene blue solution. Control +: Buffer+ laccase from T. versicolor, methylene blue solution.
351
Figure 7: Assay for ligninolytic activity (based on methylene blue decolourisation) in spent culture supernatants after incubation of T. harzianum in YNB with different amounts of CIMV lignin: A)
0.25 % (w/v), B) 0.5 % (w/v), C) 1 % (w/v), D) 2 % (w/v) for 10 days at pH 7 and 28 oC. Test: Buffer+ spent culture supernatant + methylene blue solution. Control –: (1) Buffer+ methylene blue
solution, (2): Buffer+ YNB (no enzyme), methylene blue solution. Control +: Buffer+ laccase from T. versicolor, methylene blue solution.
352
Figure 8: Growth of P. putida, P. saccharophila, P. fluorescens on M9 solid-based media supplemented with 0.25 % (w/v) of CIMV lignin, after 10 days of incubation at pH 7 and 30 0 C. Test:
inoculated plate, Control –: M9 + agar and inoculum, Control +: YNB+ glucose 0.25 % (w/v) + agar and inoculum.
353
Figure 9: Assay for ligninolytic enzyme production by P. saccharophila (on the left), P. putida (in the middle) and P. fluorescens (on the right) on M9 solid media supplemented with methylene
blue (0.05 g/) at pH 7, after 10 days of incubation at 30 oC. Test: inoculated plates, Control -: uninoculated plate.
354
Figure 10: Assay for ligninolytic enzymes by P. saccharophila (at the top), P. putida (at the bottom) and P. fluorescens (in the the middle) on M9 solid media supplemented with methylene blue
(0.05 g/) and CIMV lignin (0.25 % (w/v) at pH 7, after 5 days of incubation at 30 oC. Test: inoculated plates, Control -:uninoculated plate, Control + : plate supplemented with laccase from T.
versicolor.
355
Figure 11: Assay for ligninolytic enzyme production by P. saccharophila (on the right), P. putida (in the middle) and P. fluorescens (on the left) on M9 solid media supplemented with methylene
blue (0.05 g/L) and glucose (2.5 g/L) at pH 7, after 10 days of incubation at 30 oC. Test: inoculated plate, Control- : uninoculated plate.
356

Anna Suchy Thesis

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Development of processing technologies for

the generation of lignin with applications in

Anna SuchyAnna Suchy

Thesis submitted to Aberystwyth University for the degree of

Doctor of Philosophy in Biological Sciences

Dr. Ana Winters: Principal Investigator, IBERS, Aberystwyth University (alg@aber.ac.uk)

Dr. David Bryant: Synthetic Biologist, IBERS, Aberystwyth University (dgb@aber.ac.uk)

I would like to begin by expressing my deepest gratitude to my main supervisor Dr Ana

Thesis word count......................................................................................................... ii

1.1 Introduction .................................................................................................. 2518

2.1 Abstract ........................................................................................................ 7163

CHARACTERISTICS OF LIGNIN FROM GRASS AND WOOD........................................................... 9789

3.1 Abstract ........................................................................................................ 9789

4.1 Abstract .................................................................................................... 192185

A280 Absorbance measured at 280 nm

Mn Number average molecular weight

Ministry of Economic Affairs set a goal to derive 30 % of transportation fuels from

Increased production of second-generation biofuels using lignocellulosic biorefinery

Lignocellulosic biomass represents one of the world’s largest renewable biochemical

Lignocellulose consists mainly of cellulose, hemicellulose and lignin. However, some

Lignocellulosic biomass can be provided in two forms: as a crop or as a residue.

1.2.4.2 Lignin structure

1.2.4.3 Physical properties of lignin

1.3 LIGNIN ISOLATION

Theoretically, lignin can be isolated by different extraction methods (Zabaleta, 2012).

1.3.1.1 Braun’s lignin

1.3.1.2 Milled wood lignin

1.3.3 LIGNIN FROM BIOMASS CONVERSION PROCESSES FOR BIOREFINING

Pre-treatment is an important, initial step in a biorefinery operation (Zakzeski et al.,

Steam explosion lignin

Dilute acid pre-treatment typically employs 0.4 % - 2 % H2SO4 at temperatures of 160 oC

1.4 APPLICATIONS OF LIGNIN IN INDUSTRY

1.4.1 NEAR –TERM OPPORTUNITIES: POWER, FUEL AND SYNGAS PRODUCTS

The main aim of near-term opportunities for lignocellulosic biorefineries, is to convert

1.4.2 MEDIUM-TERM OPPORTUNITIES: MACROMOLECULES

The main aim of medium-term opportunities is to maintain the macromolecule structure

1.4.2.1 Carbon fibres

1.4.2.2 Polymer modifers

1.4.2.3 Resins/Adhesives and binders

1.4.3 LONG-TERM OPPORTUNITIES: LOW MOLECULAR WEIGHT AROMATIC OR PHENOLIC COMPOUNDS

A number of different conversion methods have been proposed to depolymerize lignin

1.5 VALORISATION OF LIGNIN

1.5.2 FUTURE MARKETS

1.6 BIO-BASED PRODUCTS- OPTIMISATION OF LIGNIN PRESCURSORS THROUGH PHYSICO-

Currently, physico-chemical modification of lignin appears to be one of the potential

Ultrafiltration (lignin purification)

Lignin solubility in organic solvents

Attempts for a copolymerization process have been conducted by researchers from

1.6.2 LIGNIN MODIFICATION/DEGRADATION BY MICROORGANISMS AND THEIR ENZYMES

1.6.2.1 Lignin degradation by fungi

1.6.2.1.1 White-rot fungi

Classes of ligninolytic enzymes:

Lignin peroxidase (LiP): Lignin peroxidase, mostly produced by Phanerochaete

Manganase Peroxidase (MnP): Manganese peroxidase (MnP) is a heme-containing

as a diffusible oxidant. It is able to oxidize secondary substrates at a distance away from

In addition to ligninases, other extracellular enzymes which act as accessory enzymes

1.6.2.1.3 Soft-rot fungi

Vanilin dehydrogenase: This is a critical enzyme for the degradation of lignin-derived

Cα-Dehydrogenase: This enzyme participates in oxidation of the α-carbon to α-carbonyl

Dioxygenase: This enzyme is a non-heme iron-dependent enzyme and catalyses the

Decarboxylase: This enzyme converts hydroxycinnamic acids into the corresponding 4-

Organism Enzyme Effect Cellular location References

Sphingobium β-etherase Catalyzes the cleavage of extracellular Gellerstedt, (2009),