Valorization of Kraft Lignin by Fractionation and Chemical Modifications for Different Applications

Selda Aminzadeh Doctoral Thesis

Wallenberg Wood Science Center (WWSC) Department of Fiber and Polymer Technology School of Engineering Sciences in Chemistry, Biotechnology and Health KTH Royal Institute of Technology Stockholm, Sweden 14th December 2018, Stockholm, Sweden

I Principal Supervisor Prof. Mikael Lindström

Co-supervisors Assoc.Prof. Olena Sevastyanova Prof. Gunnar Henriksson

ISBN: 978-91-7873-046-9 TRITA-CBH-FOU-2018-61 ISSN:1654-1081 Tryck: US-AB, Stockholm 2018

Akademisk avhandling som med tillstånd av Kungliga Tekniska Högskolan framläggs till offentlig granskning för avläggande av teknisk doktorsexamen i fiber och polymerteknologi fredagen den 14:e december 2018, Lindstedtsvägen 26, Stockholm, kl. 14:00 i sal F3. Avhandlingen försvaras på engelska. Opponent: Professor Hasan Jameel, North Carolina State University, USA

“Dedicated to my mother and father.”

III Abstract

Lignin is one of the most abundant biopolymers. Approximately 70 million tons of technical lignin is generated annually, but only little is used for products other than energy. The complexity of lignin hinders full utilization in high-value products and materials. In spite of the large recent progress of knowledge of lignin structure and biosynthesis, much is still not fully understood, including structural inhomogeneity. We made synthetic lignin at different pH’s and obtained structural differences that might explain the structural inhomogeneity of lignin.

Technical lignins from the chemical pulping are available in large scale, but the processes result in alterations, such as oxidation and condensation. Therefore, to utilize technical lignin, modifications, such as fractionation and/or chemical modifications are necessary. Fractionation with ceramic membranes is one way to lower the polydispersity of lignin. The main advantage is their tolerance towards high temperature and harsh conditions. We demonstrated that low Mw lignin was extracted from industrially produced LignoBoost lignin aiming: i) to investigate the performance of the membrane over time; ii) to analyze the antioxidant properties of the low Mw lignin.

Chemical modification can also improve the properties of lignin. By adding moieties, different properties can be obtained. Amination and methacrylation of kraft lignin were performed, as well as lignin-silica hybrid materials with potential for the adsorption were produced and investigated.

Non-modified and methacrylated lignin were used to synthesize lignin-St-DVB porous microspheres to be utilized as a sorbent for organic pollutants. The possibility to substitute styrene with methacrylated lignin was evaluated, demonstrating that interaction between lignin and DVB, and porosity increased.

Lignin has certain antibacterial properties. Un-modified and modified (aminated) lignin samples and sphere nanoparticles of lignin were tested for

their effect against gram-positive and gram-negative bacteria’s and an injectable hydrogel was developed with encapsulated lignin for being used as an injectable gel for the open wounds. Results demonstrated promising antibacterial efficiency of lignins against gram-positive, more especially better inhibition with aminated lignins against gram-positive and negative bacterium.

V SAMMANFATTNING

Lignin är en av de mest förkommande biopolymererna och ca 70 miljoner ton av tekniskt lignin produceras årligen, men endast en mindre del används till andra applikationer än energiproduktion. Ett hinder för användning av lignin i mer komplexa produkter och material är dess komplexa struktur. Trotts senare års stora framsteg när det gäller kunskap om ligninets struktur, är mycket alltjämt dåligt förstått, exempelvis angående den strukturella inhomogeniteten hos lignin. Vi har studerat detta genom att göra syntetiskt lignin vid olika pH, och erhöll strukturella skillnader som kan vara en förklaring till den strukturella inhomogeniteten.

Tekniska ligniner från kemisk massatillverkning är tillgängliga i stor skala, men processerna resulterar i strukturella modifieringar hos ligninet, såsom oxidationer och kondensationer. Därför är fraktionering och modifiering av tekniskt lignin lämpligt. Fraktionering med hjälp av keramiska membran är ett sätt att minska polydisperiteten hos lignin. Den största fördelen är membranens stora tolerans mot höga temperatur och aggressiva kemikalier. Vi använde filtrering på keramiska membran för att framställa lågmolekylärt lignin från den industriella kvaliteten LignoBoost, för att utvärdera membranens prestanda över tid, och analysera antioxidantegenskaperna hos det lågmolekylära ligninet.

Kemisk modifiering kan också användas för att förbättra egenskaperna hos lignin. Genom att koppla på grupper, kan egenskaperna ändras. Amidering och metakrylering av sulfatlignin utfördes liksom tillverkning av lignin-silikon- hybridmaterial, med potential för adsorption, och materialen undersöktes.

Omodifierat och metakrylerat lignin användes tillsammans med styren för att syntetisera porösa mikrosfärer som testades som absorbent för organiska föroreningar. Utvärdering visade att metakrylering ökade interaktionen mellan lignin och polystyren och ökade porositeten.

Lignin har viss antimikrobiell aktivitet. Omodifierade och amiderade ligninprover och sfäriska nanopartiklar av lignin testades för sin verkan mot gram-positiva och gram-negativa bakterier. Resultaten visade lovande resultat för antimikrobiell aktivitet, och särskilt för amiderade ligniner när det gäller grampositiva bakterier. En injicerbar hydrogel med inkapslat lignin utvecklades också för behandling av öppna sår.

VII List of papers

This thesis is a summary of following publications:

Paper I: A possible explanation for the structural inhomogeneity of lignin in LCC networks. Aminzadeh. S., Zhang. L., Henriksson. G. Wood Sci Techn., 2017, 51, 1365- 1376. DOI: 10.1007/s00226-017-0941-6

Paper II: Membrane filtration of kraft lignin: Structural characteristics and antioxidant activity of the low-molecular-weight fraction. Aminzadeh. S., Lauberts. M., Dobele. G., Ponomarenko. J., Tuve Mattsson. T., Lindström. E. M., Sevastyanova. O. Industrial Crops & Products., 2018, 112, 200-209 DOI: 10.1016/j.indcrop.2017.11.042

Paper III: Peculiarities of synthesis and properties of lignin-silica nanocomposites prepared by sol-gel method Budnyak. T., Aminzadeh. S., Pylypchuk. I., Riazanova. A., Tertykh. V., Lindström. M., Sevastyanova. O. Accepted to Nanomaterials Journal

Paper IV: Methylene Blue dye sorption by hybrid materials from technical lignins Budnyak. T., Aminzadeh. S., Pylypchuk. I., Sternike. D.,Tertykh. V., Lindström. M., Sevastyanova. O. Journal of Environmental Chemical Engineering, 2018, 6, 4997-5007. DOI: 10.1016/j.jece.2018.07.041

Paper V: Synthesis and structure characterization of polymeric nanoporous microspheres with lignin Goliszek. M., Podkoscielna. B., Fila. K., Riazanova. V. A., Aminzadeh. S., Sevastyanova. O., Gun’ko. V. M. Cellulose, 2018. DOI: 10.1007/s10570-018- 2009-7

Paper VI: Lignin based hydrogel for the antibacterial application Aminzadeh. S., Haghniaz. R., Ottenhall. A., Sevastyanova. O., Lindström. E. M., Khademhosseini. A. (Manuscript)

VIII

The author’s contributions to the appended papers are the following:

Paper I, II: Principal author. Performed most of the experiments and most of the manuscript preparations. (Pyrolysis and antioxidant studies were made in Riga).

Paper VI: Principal author. Performed the planning and designing the project, all of the experiments and manuscript preparation.

Paper III and IV: Second author. Performed most of the experiments, participated in discussions and prepared some parts of the manuscript.

Paper V: Fourth author. Performed part of the experiments (synthesis, 13C NMR, thermal analysis and SEM), participated in discussions and partly in the preparation and revision of the manuscript.

Paper VI: First author. Performed most of the experiments and most of the manuscript preparation (design of this project, sample preparations, Instron test, cell studies, nanoparticle preparations were performed in the US, Harvard-MIT medical centre-UCLA Uni).

Conference Publications:

1. On the Crossflow Membrane Fractionation of Lignoboost Kraft Lignin - Characterization of Low Molecular Weight Fractions.

Aminzadeh. S., Sevastyanaova. O., Mattsson. T., Lindström. M. 251th ACS National Meeting, Oral Proceeding, 13th March, 2016, San Diego, USA.

2. Membrane Filtration of LignoBoost Kraft Lignin: Fouling Phenomena and Properties of the Low Molecular Weight Fraction.

Aminzadeh, S., Lauberts, M., Dobele, G., Ponomarenko, J., Mattsson, T., Sevastyanova, O., Lindström, M.E., Nordic Wood Biorefinery Conference (NWBC), Poster Proceedings pp. 220-222, March 28th-30th, 2017, Stockholm, Sweden.

IX 3. Membrane Filtration of LignoBoost Kraft Lignin: Properties of the Low Molecular Weight Fraction Aminzadeh, S., Lauberts, M., Dobele, G., Ponomarenko, J., Mattsson, T., Sevastyanova, O., Lindström, M.E.: 19th International Symposium on Wood Fibre and Polymer Chemistry (ISWFPC), Oral Proceedings pp. 246-250, August 31th – September 1st, 2017, Porto Seguro, Brazil;

4. Novel Lignin/Silica Hybrids: Peculiarities of Synthesis and Application

Budnyak, T.M., Aminzadeh, S., Pylypchuk, I.V., Riazanova, A.V., Lindström, M.E., Sevastyanova, O. 15th European Workshop on Lignocellulosics and Pulp (EWLP), Poster Proceeding pp. 107-110, June 26th-29th, 2018, Aveiro, Port

5. Valorization of Kraft lignin by fractionation and chemical modification for different applications Aminzadeh. S., Sevastyanova. O., Henriksson. G., Lindström. M. Marcus Wallenberg Prize conference, Poster and Pitch proceeding, 23th-26th September, 2018, Stockholm, Sweden.

Abbreviations

LBL: LignoBoost Lignin CFBL: Clean Flow Black Lignin LMw: Low Molecular Weight DHP: Dehydrogenase polymer H: para-Hydroxyphenyl unit G: Guaiacyl unit S: Syringyl unit LCC: Lignin Carbohydrate Complex NMR: Nuclear Magnetic Resonance Spectroscopy HSQC: Hetronuclear Single Quantum Coherence Spectroscopy 31P-NMR: 31Phosphorus Nuclear Magnetic Resonance Spectroscopy 13C-NMR: 13Carbon Nuclear Magnetic Resonance Spectroscopy DMSO: Dimethyl Sulfoxide APTES: 3-Aminopropyltriethoxysilane TEOS: 1-1-Tetraethoxysilane SEC: Size Exclusion Chromatography PS: Poly-dispersity CFF: Cross Flow Filtration FTIR: Fourier Transform Infrared Spectroscopy SEM: Scanning Electron Microscopy DLS: Dynamic Light Scattering LP: Laponite (Silicate Nanoplatelets) St: Styrene

XI DVD: Divinylbenzene MMA: Methyl Methacrylate PHEMT: Poly-2 Hydroxyehyl methacrylate PGEMA: Poly- Glucosylethyl Methacrylate

XII

Table of Contents

1. INTRODUCTION ...... 1

1-1 BACKGROUND ...... 1 1-2 LIGNIN AND ITS PROPERTIES ...... 2 1-3 LIGNIN ISOLATION ...... 5 1-3-1 Kraft Process ...... 5 1-3-2 Sulfite process ...... 6 1-3-3 Soda (Alkaline) process ...... 8 1-3-4 Organosolv processes ...... 8 1-4 FRACTIONATION OF KRAFT BLACK LIQUOR ...... 9 1-4-1 Selective precipitation of lignin ...... 10 1-4-2 LignoBoost Kraft lignin ...... 11 1-4-3 Cross-Flow filtration ...... 12 1-5 VALORIZATION OF LIGNIN ...... 13 1-5-1 Uses of non-chemically-modified lignin (the antioxidant activity of lignin) ...... 14 1-5-2 Uses of chemically-modified lignin ...... 15 1-6 AIM OF THIS STUDY ...... 16

2. MATERIALS AND METHODS ...... 18

2-1 MATERIALS ...... 18 2-2 METHODS ...... 19 2-3 ANALYSIS ...... 22

3. RESULTS AND DISCUSSION ...... 28

3-1 EFFECT OF MICROENVIRONMENT ON THE SYNTHESIZED LIGNIN’S STRUCTURE (PAPER I) .... 28 3-2 FRACTIONATION BY ULTRAFILTRATION (PAPER II) ...... 31 3-2-1 Impact of time on the membrane performance ...... 31 3-2-2 Characterization of Low Mw lignin ...... 35

XIII 3-3 CHEMICAL MODIFICATION OF TECHNICAL LIGNINS (PAPER III, IV, V, VI) ...... 39 3-3-1 Physico-Chemical properties of technical lignins (LBL & CFBL) ...... 39 3-3-2 Amination of Technical Lignins (Paper III, IV, VI) ...... 43 3-3-3 Methacrylation of lignin (Paper V) ...... 46 3-4 LIGNIN MATERIALS (PAPER III, IV, V, VI) ...... 48 3-4-1 Lignin-Si hybrid materials (Paper III, IV) ...... 48 3-4-2 Lignin-St-DVB Porous microsphere (Paper V) ...... 54 3-4-3 Lignin nanoparticles (Paper VI) ...... 57 3-4-4 Lignin hydrogels (Paper VI) ...... 59 3-5 APPLICATION OF LIGNIN MATERIALS...... 61 3-5-1 Antioxidants (Paper II) ...... 61 3-5-2 Sorbents (Paper III, IV) ...... 64 3-5-3 Antibacterial (Paper VI) ...... 67

4. CONCLUSIONS ...... 74

5. FUTURE OUTLOOK...... 76

6. ACKNOWLEDGEMENTS ...... 77

7. REFERENCES ...... 79

XIV

1. Introduction 1-1 Background

There are numerous problems associated with fossil fuel consumption, i.e. the source depletion, the elevated production of greenhouse gas and environmental issues. Moreover, with higher fossil fuel consumption and consequently the higher amount of generated greenhouse gas, the CO2 concentration goes up which results in global warming. All these problems, emphasizes the special need for the replacement of fossil fuel by natural, biodegradable and sustainable resources.1, 2, 3, 4

Lately, there has been a stronger focus to develop polymers instead of traditional metals and glass-based materials due to their easy processing, low cost and availability.5, 6, 7, 2 However, because of the mentioned problems associated with the use of fossil fuel, there is growing attention for bio- renewable resources including cellulose, polysaccharides, lignin, alginate and animal protein-based biopolymers, etc.5, 2

In this respect, replacing of fossil-based carbon source with the plant-based (i.e., biomass) carbon source will favor towards a greener environment, more specifically, in a long-term climate goal.8, 9 The concept of biorefinery simply meaning the use of biomass instead of oil for producing energy and chemicals. 10, 11 Forest industry is one of the key sources which can fulfil the needs of the bio-based economy in the different areas. The extraction and conversion of biomass to energy, high-value chemicals and plastics are the primary goals from biorefinery perspective.

————— Introduction —————

In Sweden, the forest industry is one of the largest production industries and has a significant contribution to Sweden’s export income.12 Although in general, pulp and industry is facing some problems due to lower demand for traditional products, like books and newspapers because of a massive competition from the digital world. There is a growing demand for packaging, hygienic products and textile. Besides, the necessary need to turn from fossil fuel source to bio-based opens up a special place for forest products, e.g., wood, cellulose, hemicellulose and lignin. The implementation of these components in high-value products demands in-depth studies concerning sample separations and characterizations. There has been an enormous number of publications.1, 8, 13,1, 14, 15, 16 over the last years to better understand wood components’ structures and properties to make them compatible with oil-based products.

1-2 Lignin and its properties

Lignin is the most abundant biopolymer after cellulose, and the first abundant aromatic biomass on the earth.13, 8 There are three main aromatic alcohols (polypropane units), including p-coumaryl, coniferyl alcohol and sinapyl alcohol which construct lignin’s structure through radical polymerization. These monolignols are called p-hydroxyphenyl (H, from coumaryl alcohol), guaiacyl (G, from coniferyl alcohol) and syringyl (S, from sinapyl alcohol). The main difference between these units is the number of methoxy groups attaching to the aromatic ring as shows at Fig.1.13, 17, 18

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Figure 1. Three major monomers of lignin forming the lignin polymer

Native lignin, in general, can be described as a three dimensional and amorphous polymer; however, it does not have the same unique and well- defined structures in all the plants. 19 Based on the lignocellulosic biomass, it can have different monolignols dominance. Therefore, the structure of lignin can vary corresponding to the type of biomass, external conditions during growth such as climate and seasonal changes. Lignin can be found in different ratios based on the biomass type with values around 25-35% in softwood and 15-25% in hardwood. This value is much less in the grass and annual plants around 10% and 3% respectively. In respect to the biomass sources, softwood lignins are mainly composed of G-unit, whereas, hardwood lignin is a mixture of S and G units, however, containing the higher proportion of S-unit. P- hydroxyphenyl unit (H) is found in a small amount in both softwood and hardwood lignins but substantially presents in non-woody species such as grass. 13, 8, 20

Lignin is composed of different units which are connected together through various inter-unit linkages, e.g., ether linkages (C-O-C) or carbon-carbon bonds (C-C). These inter-unit linkages impart heterogeneous complex structures to the

3 ————— Introduction —————

lignin. The dominant is β–O-4 ether linkage ( 45-60%) which is followed by other types of linkages such as α–O-4, β-5, 5-5´, 4-O-5 and β-β´. 13, 21, 22, 23

As shown by many studies, there are different types of functional groups in lignin, i.e., aromatic methoxy groups, aliphatic and aromatic hydroxyl, carboxyl and a small amount of carbonyl groups.24, 25, 26 There has been discussions13, 27, 28 about lignin structures differences and some parameters which can affect lignin’s structure. For instance, Henriksson 2018 28 proposing some hypothesis that how the type of hemicellulose can affect lignin’s structure. It has been discussed that due to the presence of carboxylic acid on glucuronic acid side groups of xylan might create locally acidic environment close to xylan side, which simply means the presence of extra protons. As depicted in Scheme 1 (Results and Discussions part), the path for creating β-O-4 demands protonation, therefore, this acid microenvironment favors the creation of more β-O-4. Henriksson 2018 discusses that if the presence of the certain type of hemicellulose gives different lignin structure, then these different hemicelluloses can direct the structure of lignin in plant cell wall. As he discussed, based on the mentioned hypothesis, lignin bounds to glucomannan have more condensed structure and more ‘‘web-shaped’’ structure, whereas, lignin bounds to xylan is expected to be more homogenous and linear. These types of xylan-bound lignin should have more LCC bounds since quinine methide intermediate is created during β-O-4 formation. In addition, other parameters such as the presence of various types of monolignols with different degree of methoxylation and the overall pH of the cell wall are very determining.

————— Introduction —————

1-3 Lignin Isolation

Nowadays, there are two ways mainly to produce chemical pulp; kraft pulping (also known as sulphate process in Sweden) which is dominating and the sulfite process. Both processes generate a huge amount of technical lignin production as a by-product.29, 17, 13 The main goal of these processes are to extract cellulose (and also hemicelluloses) with the employment of chemicals at high temperature. The extracted cellulose is considerably used for the production of paper in addition to being utilized for the other high-value materials. In spite of higher lignin production as a by-product, lignin has never been considered for the valorization and application in value-added products over the past years and it has mainly been burned for the energy production as fuel.30

There are two other types of pulp processes, soda pulping and organosolv process which result in the production of sulfur-free lignins.29, 31, 32, 33 ; however the processes are limited in use.

1-3-1 Kraft Process

Kraft process is the main process which is applied in a large scale production (85% of world production). The production of kraft lignin started to surpass sulfite pulps since the 1950s due to some benefits, i.e., easier and economical ways of the recovery of cooking chemicals and giving better pulp quality plus easier to scale up.13 In this process, wood chips are digested in the alkaline cooking liquor including, sodium hydroxide (NaOH) and sodium sulfide

(Na2S), also named as white liquor. Wood chips are cooked by using this white

5 ————— Introduction —————

liquor at pH>13 and temperature between 150-170 ºC. This process ends up with the separated and disintegrated cellulose fibers and black liquor which contains degraded lignin, degraded carbohydrates, extractives and some inorganic materials coming from wood and cooking chemicals. In weak black liquor (before evaporation) the dry content is approximately 15-20% after kraft cooking. 13 Although, native lignin in biomass is a branched macromolecule with a high molecular weight, lignin in black liquor consist of degraded and chemically modified fragments having high polydispersity.1, 34 It has been shown by various studies that the lignin presents in black liquor (technical lignin) has a more condensed and heterogeneous structure than native lignin with respect to molecular weight and functionality, i.e., less aliphatic, more hydroxyl and very rich in phenolic groups.35, 36 Generally, the generated kraft lignin is exclusively burned for energy production to cover the energy demand of the kraft pulp process.1

1-3-2 Sulfite process

Sulfite process was invented before kraft process; however, it was less used lately due to some advanatge of kraft pulp. It was discovered by B. Tilghman in 1866 when he patented a method for the pulp extraction by using aqueous solutions of calcium hydrogen sulfite and sulfur dioxide. One decade after, C. D. Ekman initiated to use this method for the production in Sweden, basically the first official use of this method started by him in Sweden. This method was dominant until 1950s when the kraft process started to surpass the position.13

————— Introduction —————

The sulfite process is based on the implementation of an aqueous solution of chemicals such as sulfur dioxide (SO2) and inorganic bases (counter ions), i.e., calcium, sodium, magnesium or ammonium. In this process, a high amount of sulfur is used during the cooking process that results in the formation of a 18 sulfonate group (SO3) on the aliphatic side chains. The presence of sulfonate groups makes this type of extracted lignins soluble in water and insoluble in organic solvents.30

Lignosulfonates are the main crude component of lignin which can be used for high-value application instead of simply being burned for energy production. For instance, it has been used in the building sector as a concrete additive to provide plasticity and flowability to the concrete. It has also been used as a binder in animal feed pellets.24, 30, 37 Lignosulfonate is used for the production of different materials, mainly vanillin. Presence of high density of functional group in lignosulfonates gives unique colloidal properties to this type of lignin which can be utilized as stabilizer, emulsifiers, dispersing agents, surfactant and adhesives. In addition, lignosulfonate can be used in dust control, oil well drilling, road building, pesticides for agriculture application and cleaning and decontamination agent in water and soils. 31, 24, 37 There are few examples of the front-runner companies in this field, utilizing lignosulfonate, i.e., Borregaard company located in Norway (and having activities in some other places) as well as Nippon Paper in Japan.

7 ————— Introduction —————

1-3-3 Soda (Alkaline) process

This method was invented at 1845 as a first chemical process; however, it is very effective method only for biomass with lesser lignin content than in wood. Therefore, nowadays it is mainly being used for pulping of the annual plants and agricultural residues.31 This process is based on degrading of biomass by using sodium hydroxide at elevated temperature and pressure. The privilege of this method as being sulfur-free process that generates sulfur-free lignin and also has more potential for the application in high value products. Moreover, in some studies, it has been shown that soda lignin was used for the production of polyphenol (PF) resins as a replacement for the phenol which comes from petroleum-based source.25, 26, 38

1-3-4 Organosolv processes

Organosolv processes use various organic solvent, e.g., ethanol, methanol, acetic acid, etc to solubilize lignin and hemicelluloses. There are a couple of advantages regarding using organosolv processes that bring special attention for these technique and the lignin obtained from them. Firstly, because of using low boiling point solvents such as methanol and ethanol, and some other types of organic acids, e.g., formic and acetic acid, the solvent can be recovered through distillation without creating too much pollution.24 Secondly, it offers sulfur free lignin as a by-product which can have better properties for the further high- value applications. This type of lignin is ash and sulfur-free and better solubility in organic solvents than kraft and soda lignins.39 These pulping have been used recently in various biorefinery processes, where the cellulose part is used for

————— Introduction —————

the production of ethanol. In this case, the use of lignin in value-added applications is necessary for the economics of the process.

The first efficient organosolv process based on the applying of ethanol/water was Alcell® process in the early 1970s. Later, it was modified and dominated by Lignol company located in Canada.18, 31

1-4 Fractionation of kraft black liquor

Lignin’s structure is changed after the pulping process mainly because of the usage of harsh chemicals at high temperature.35 This results in some new chemical reactions which lead to the degradation and formation of more condensed lignin structures. Since position in aromatic carbon 5 region is free in native lignin mainly in softwood lignin, therefore there is a higher chance of the formation of new condensed linkages for softwood lignin. All of these have a negative impact in a way that native lignin structure is entirely different than technical lignin.35, 36, 40, 1, 24, 18, 26 The proportion and availability of these functional group are determined by the type of biomass in addition to the separation processes. These functional groups are very important for lignin’s characterizations as well as further modification of lignin regarding making new and up-graded chemicals and products. Though, few percentages of these functional groups, especially hydroxyl groups are available in technical lignin mainly because he condensed inter-unit linkages occupy most of them. Additionally, lignin has linkages to the carbohydrate fragments including arabinose, xylose and galactose through lignin-carbohydrate linkages (LCC).41, 42, 43 Consequently, in spite of many years intensive studies regarding lignins

9 ————— Introduction —————

structures and the content of functional groups, still there is a lack of complete understanding of all type of structures. It has been reported that a large amount of technical lignin, approximately 50-70 million tons of lignin are produced annually.8 In comparison with this mass production of technical lignin as a by- product from pulp and paper industry, only a few percentages of lignosulfonate lignin are utilized for high-value applications.38

To utilize lignin in a very efficient way, fractionation of lignin plays a significant role. During last year’s, there have been many studies which demonstrated how fractionation of lignin improves their properties, e.g., improved polydispersity, more homogeneous functionality and chemical properties. Low Mw lignin, for example, is very rich in phenolic content. Such lignin materials have very interesting potentials, i.e., the possibility of further modifications for different applications and high antioxidant activities.44, 45, 46, 47 To understand structure and properties of low Mw lignin, there have been some studies regarding the extraction of low Mw lignin44, 48, 49, 47; however, the efficient and more convenient method is yet to be found.

Fractionation of lignin can be achieved in different ways. Acid precipitation50, 51, organic solvent fractionation34, 39, 9, 52 and membrane fractionation are different ways of the extraction of lignin.38, 44, 53, 47, 49, 54, 55, 56

1-4-1 Selective precipitation of lignin

To utilize lignin as a by-product from pulping processes lignin should be precipitated and extracted. In general, lignin is soluble in black liquor due to the

————— Introduction —————

deprotonation of functional groups. Therefore, by introducing the H+ as a proton donator, it can gradually start to precipitate.13, 44, 53 The precipitation factor can be affected by pKa value. Though, this pKa values can vary depending on the temperatures, ionic strength, molecular weight as well as the dry content of black liquor.57, 58, 59, 60 There were two main methods for the precipitation of lignin from black liquor. Initially, it started in 1872, a method invented by Tessie du Motay61, in which carbon dioxide was used for the precipitation of impurities to end up with pure liquor that could be re-caustified and reused. Moreover, later other scientist studied the use of carbon dioxide as well as using acid for the precipitation of lignin in a more practical way. The precipitation step was performed at ahigher temperature.62, 63, 64, 65, 66, 67

1-4-2 LignoBoost Kraft lignin

LignoBoost lignin is a recently developed process68, in which there is a displacement washing step where lignin with higher purity can be obtained. After precipitation, the material is filtered and the filter cake is redispersed at low pH (2-4). The suspension that is formed can easily be re-filtered and washed by displacement washing. There are some valuable benefits in the development of the LignoBoost lignin process. LignoBoost lignin is rather pure lignin which can be used for further applications. Only 0.2-1% ash content and 1.5-2.8% carbohydrates have been reported as impurities. More importantly, by utilizing LignoBoost lignin, the load in recovery boiler can be reduced which is a bottleneck in the pulp mills.68, 69, 70

11 ————— Introduction —————

1-4-3 Cross-Flow filtration

Membrane separation can be considered as a very useful, efficient and versatile technique which can be used in many different areas, e.g., the food industry, water purification and the pharmaceutical industry.49, 71, 72, 73, 74, 75 In respect to the separation of lignin, the effect of different parameters during separation, i.e., concentration, temperature and cost of the lignin separation by ultrafiltration technique were investigated.76, 77

There are some advantages regarding implementing the ceramic membrane in comparison to other techniques. The main advantage of filtration with ceramic membranes is the direct installation and withdrawing of the sample from black liquor without pH and temperature adjustment.78 The partial lignin extraction (depending on the Mw cut-off of the used membrane) will have a significant impact on the efficiency of recovery boiler in a way that by the extraction of lignin the liquor load can get reduced in recovery boiler which is a bottleneck in industry.78, 79, 80 Consequently, this will have a positive impact on the pulp production capacity.

By using the ceramic membrane with different cut-offs, lignin with more well- defined molecular weight, lower polydispersity and eventually tailored properties can be achieved.44, 47

The Cross-flow filtration (CFF) technique was applied in this project. CFF is a dynamic filtration which particles or molecules can be separated based on the size and shape. This technique works in a circulation mode where the liquid sample flows parallel to the filter media and start to penetrate through the

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membrane based on the transmembrane pressure (TMP). The turbulent flow which can be adjusted based on the applied pressure and flow is desirable in this type of extraction to reduce the build-up of particles and molecules on the membrane surface.44, 53

1-5 Valorization of lignin

Lignin as a poly-phenolic component with some outstanding properties can potentially be utilized in many different products. It has carbon-rich content, in addition to the presence of aromatic and aliphatic units with different functional groups which make it very suitable for many chemical conversion.2 More importantly, this material is the cheap by-product and produced in a large scale in pulp and paper and biorefinery industries. Therefore, intense scientific studies are required to understand better the lignin structures, processing- properties relationship to valorize it in a more efficient way.29

Lignin has been studied for its usage in different applications, e.g., in the production of vanillin81 and phenols; as a dispersant in cement and gypsum blends82, 83, an emulsifier84, 85 , an adsorbent85, 86, 87 , a carbon fiber precursor88, 89, 90, and an antioxidant44, 91, and co-reagent in phenol-formaldehyde resins55, 92, 93, 94 and thermoplastics synthesis.95

In some products, lignin can be used directly without any chemical modifications, for others improved chemical reactivity is required.18, 9 A general scheme for the various types of uses of technical lignins is shown in Fig. 2.

13 ————— Introduction —————

Fig. 2 Global scheme of the uses of lignin with or without chemical modifications 18 ; (re-drawn with permission of Elsevier).

1-5-1 Uses of non-chemically-modified lignin (the antioxidant activity of lignin)

Lignin as a polyphenol substance, perform very promising antioxidant properties44, 96, 97, 98. This property can be explained mainly due to the presence of phenolic-hydroxyl group which prevent oxidation reaction as well as scavenge free radicals through stabilization of free radical by the phenolic group. Presences of different functional groups have a different impact on the lignin’s antioxidant properties. For instance, methoxy group increases this effect, whereas, double bond in aliphatic unit has an opposite impact. Despite some studies which demonstrated the potential of lignin as an antioxidant, the application in an industrial scale is yet to be developed.44, 98, 99, 100, 101

————— Introduction —————

1-5-2 Uses of chemically-modified lignin

Lots of studies by different scientists showed the great potential of lignin, especially lignin driven composite materials for the adsorption of dyes and inorganic contaminants. Lignin, with its aromatic structure in addition to the presence of a large number of functional hydroxyl groups, has great potential as a sorbent.50, 102, 103 Recently there has been many studies by different scientists concerning the chemical modification of lignin for different applications. For instance, Dan Kai et al. used methyl methacrylate (MMA) to modify lignin and produced highly stretchable lignin-based electrospun nanofibers for the potential biomedical application.104 Moreover, the lignin- based hydrogel can be produced through the introduction of the different chemicals including poly-2-hydroxyethyl methacrylate (PHEMT), poly- glucosylethyl Methacrylate( PGEMA), etc. These type of hydrogels can be utilized in various areas, e.g., water purification, drug delivery, biomedical applications and biomimetic scaffolds.105 Lignin-St-DVB composite was developed mainly with the purpose of having the potential for the adsorption applications. It was proved that how chemical modification of lignin with methacryloyl chloride can enhance the reactivity of lignin.87, 106 Additionally, there are studies in respect to the antibacterial properties of lignin against gram-positive and yeast, whereas, almost all the studies showing no antibacterial activity against gram-negative. The main antibacterial property of lignin was attributed to the polyphenolic structure of lignin. These phenyl groups can damage the cell membranes of microorganism. In our study in the

15 ————— Introduction —————

last part of work it is demonstrated that how the modification of lignin can enhance its antibacterial properties against gram-negative as well.91, 107, 108, 109

1-6 Aim of this study

The main purpose of this study was to study the distinct properties of unfractionated (initial) and fractionated technical lignins and to try to utilize them in a modified and unmodified form for different applications. To achieve these goals, different techniques including 2D HSQC NMR, 31P NMR, SEC, TGA and pyrolysis-GC/FID were used to characterize mentioned lignins properties. After understanding the structure of lignin, the radical scavenging properties of lignins were analyzed in an unmodified form to understand their potential for the antioxidant applications. Also, lignin was modified in different ways for the different application, i.e., construction of porous lignin-hybrid materials as a sorbent and making lignin-based injectable hydrogels for the antibacterial purposes. Therefore, the main objectives of this thesis are based on the following purposes:

1. Fundamental studies with respect to the lignin structures diversity in the different microenvironment (Paper I). 2. Cross-flow membrane fractionation of technical lignin, extraction of low Mw lignin with rich phenolic content-studying the antioxidant properties of low Mw and their future potential (Paper II).

————— Introduction —————

3. Making mesoporous lignin-silica sorbent, characterization studies and sorbents’ technical potential for the adsorption of methylene blue dye (Paper III & IV). 4. Formation of lignin based sphere particles with the adsorption capacity; the main purpose was the replacement of styrene with lignin which was driven from the sustainable and eco-friendly sources (Paper V). 5. Chemical modifications and development of lignin based injectable hydrogels for the antibacterial applications (Paper VI)

17 ————— Materials and Methods —————

2. Materials and Methods 2-1 Materials

The LignoBoost Kraft lignin powder used in this study was produced from Nordic softwood and kindly supplied from a plant in northern Europe. Clean Flow Black lignin (CFBL) was low molecular weight lignin (LMw) produced by ultrafiltration technique equipped with the ceramic membrane which was kindly supplied by the Clean Flow Black company located in Sweden.

Sodium hydroxide (Sigma Aldrich, Germany), sulfuric acid (>95%) (Fisher Scientific, UK), Sodium chloride (Sigma Aldrich, Germany) were bought commercially. Ultrasil 10 was purchased from Ecolab AB (Alvsjö, Stockholm, Sweden). Other chemicals including, coniferyl aldehyde (4-Hydroxy-3- methoxycinnamaldehyde, Sigma Aldrich), horseradish peroxidase (HRP, 250-

330 units/mg, Sigma Aldrich), hydrogen peroxide solution (30% wt in H2O, Sigma Aldrich), sodium borohydride (Sigma Aldrich) and ethyl acetate (> 99.5%, Sigma Aldrich), 3-Aminopropyltriethoxysilane (APTES), tetraethoxysilane (TEOS), 1,4-dioxane and formaldehyde were purchased (Sigma-Aldrich), ethanol (VWR) were provided from above mentioned companies. All the chemicals used were of analytical grades.

Dimethyl sulfoxide anhydrous was obtained from Sigma–Aldrich (MO, USA). NIH/3T3 mouse fibroblasts cell lines were procured from the American Type Culture Collection (ATCC, VA, USA). Dulbecco’s modified Eagle medium (DMEM, 4.5 g/L glucose), Fetal bovine serum (FBS), 0.25% trypsin- ethylenediaminetetraacetic acid (EDTA), penicillin/streptomycin solution (P/S,

————— Materials and Methods —————

10,000 U/mL/10,000 µg/mL), and Dulbecco's phosphate-buffered saline (DPBS, 1X) were purchased from Gibco (NY, USA). MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) labelling and detection kit, and LIVE/DEADTM viability/cytotoxicity kit were supplied from Invitrogen (OR, USA). Cells were grown in cell culture flasks (Corning, NY, USA), polystyrene 12-well or 96-well tissue culture treated plates (Falcon, NC, USA).

2-2 Methods

Synthesis of Coniferyl Alcohol

A certain amount of coniferyl aldehyde was dissolved in ethyl acetate (> 99.5%). The reduction was carried out by addition of sodium borohydride as a reductive agent, and the solution was stirred overnight at room temperature. After one overnight running, the reaction was quenched with the addition of water, and the oily product was recrystallized from dichloromethane/petroleum ether to get pale yellow powder (Paper I). H-NMR was used to verify the complete reduction of the initial sample.110

Synthetic lignin (Dehydrogenized polymer, DHP)

Synthesis of lignin (DHP) was performed as reported by other scientists 111 by utilizing horseradish peroxide (HRP) type IV. This reaction was carried out at three different pH, i.e., 3.5, 4.5 and 6.5 pH separately. For the synthesis, certain amount of synthesized coniferyl alcohol was dissolved in acetone, and mixed

19 ————— Materials and Methods —————

with 200 ml of 5 mM of the corresponding buffer to obtain solutions with a certain pH (including pH 3.5, pH 4.5, and pH 6.5) making solution A. In parallel, solution B containing approximately 1.5 ml hydrogen peroxide 30% (48.9 mmol) and solution C containing 5 mg of the enzyme Horseradish peroxidase (1250 – 1650 units) were prepared. The DHPs were made according to the “end-wise polymerization”112 in which solutions A and B were dropped in solution C at the controlled speed of 10 ml/min. The reaction was run at room temperature and the yellow pellet was collected as a product (Paper I).

Synthesis of lignin-silica hybrid composites

Lignin-silica composites were obtained by the sol-gel method. For that process, initially, 2 different types of lignins, including LBL and CFBL were modified by aminomethylation through Mannich reaction. In the next step for the sol-gel process, TEOS hydrolysis was applied to create a silica network (Paper III).

Amination of lignin with the aim of antibacterial properties

To investigate the effect of amination group on the antibacterial properties of lignin, a Mannich reaction was implemented for LignoBoost and Clean Flow Black lignins separately. Lignin samples were dissolved in 80% dioxane solvent and the reaction proceed by the addition of 5.5 mmol of 40% dimethylamine aqueous solution, 5.5 mmol of a 37% formaldehyde aqueous solution, and 0.2 ml of acetic acid. The reaction was continued at 60ºC for 4 h under stirring. The produced sample was purified by dialysis with a molecular weight cut-off of 1000 Da and then freeze-dried (Paper VI).

————— Materials and Methods —————

Methacrylation of lignin

To obtain lignin methacrylates, lignin (10 g) was subjected to the reaction either with methacryloyl chloride or with methacrylic anhydride (1:1 w/v) in dioxane (1:10 w/v). In later case, the mixture pyridine and dioxane 1:10 (v/v) was used. 0.5% hydroquinone (w/v solution) was used as an inhibitor of polymerization in both reactions113 (Paper V).

Preparation of lignin nanoparticles

Ten mg ml-1 lignin was dissolved in THF and stirred for a couple of hours to ensure proper solubility. The solution was filtered through a 0.45 μm syringe filter and introduced into a dialysis bag. (Spectra/Por® 1 Standard RC Dry Dialysis Tubing, 6–8 kDa, Spectrum Labs, USA) which was then immersed in deionized water. Water was changed 2 times a day and this procedure was run for 3 days 114 (Paper VI).

Development of lignin-silicate nanoplatelets (laponite) hydrogel

A certain amount of lignin was incorporated in silicate nanoplatelets to end up with the hydrogel with shear-thinning properties. For this purpose, separated stock solutions of each sample were prepared and mixed thoroughly through a vortex in certain ratios (Paper V).

21 ————— Materials and Methods —————

Ultrafiltration experiment

Two types of low Mw lignins were used for these projects. Clean Flow Balck lignin (CFBL) which was separated by 5000 Da ceramic membrane and provided by a clean flow black company located in Sweden (Paper III-V). In Paper II membrane cross-flow filtration experiments were performed using bench-scale membrane equipment. The membrane unit was a KeraseptTM unit purchased from Novasep, France. For this investigation, a single-channel ceramic membrane (TAMI Industries, model MSKTB 0251001, Nyons, France) with a cut off of 1000 Da was used.

2-3 Analysis

Evaluation of antioxidant properties

The DPPH● (2,2-diphenyl-1-picrylhydrazyl) and The ABTS●+ radical scavenging tests were performed as described elsewhere.115 The free-radical scavenging activity was expressed as the IC50 (concentration required for 50% inhibition of free radicals). An antioxidant activity was measured by spectrophotometrically using a PerkinElmer Lambda 650 UV/VIS spectrometer (PerkinElmer Instruments, Shelton, Connecticut, USA). The free-radical scavenging activity was also expressed as the IC50. The ORAC assay was performed according to a method described previously116 using a BioTek Synergy HT microplate reader (BioTek Instruments, Winooski, Vermont, USA). The lignin radical absorbance capacity was expressed as the Trolox equivalent, TE, per gram of lignin sample. A higher TE value indicated higher antioxidant capacity (Paper II).

————— Materials and Methods —————

Size Exclusion Chromatography (SEC/THF)

All the samples were acetylated in advance before SEC/THF measurement. After acetylation, the sample was dissolved in 1 ml of HPLC grade tetrahydrofuran (THF) and the resulting solution filtered through a 5 μm PTFE syringe filter. Size exclusion chromatography (SEC) analysis was performed using a Waters instrument system (Waters Sverige AB, Sollentuna, Sweden) consisting of a 515 HPLC-pump, 2707 autosampler and 2998 photodiode array detector (operated at 254 and 280 nm). HPLC-grade tetrahydrofuran was used as a mobile phase using a flow of 0.3 ml/min. Separation was achieved on Waters Ultrastyragel HR4, HR2 and HR0.5 4.6 x 300 mm solvent efficient columns connected in series and operated at 35°C. Calibration was performed using polystyrene standards with nominal molecular weights ranging from 480 to 176 000 Da and Waters Empower 3 build 3471 software.

Size Exclusion Chromatography (SEC)

The molecular weight of the lignin samples was characterized by size exclusion chromatography using an SEC 1260 Infinity instrument (Polymer Standard Services, Germany) coupled to a dual system detector (UV, RI). The separation system consisted of a PSS GRAM pre-column, PSS GRAM 100 Å and PSS GRAM 10 000 Å analytical columns thermostated at 60°C and connected in series. DMSO + 0.5% LiBr (w/w) was used as the mobile phase. Pullulan standards with Mw within the ranging from 708 kDa to 342 Da were used for the standard calibration.

23 ————— Materials and Methods —————

Nuclear Magnetic Resonance Spectroscopy (NMR)

31P-NMR

The content of functional groups in lignin samples was measured by 31P NMR 60. The certain amount of sample was phosphitylated using 2-chloro-4,4,5,5- tetramethyl-1,3,2-dioxaphospholane. Endo-N-hydroxy-5-norbornene-2,3- dicarboximide (e-HNDI) (Sigma Aldrich, 40 mg/mL) and chromium (Ш) acetylacetonate (Aldrich, 5 mg/mL) were used as an internal standard and relaxation reagent, respectively. CDCl3 was used to dissolve the sample prior to analysis. The 31P NMR experiment was performed with a 90° pulse angle, inverse gated proton decoupling and a delay time of 10 s. For analysis, 256 scans with a time delay of 6 s and a total runtime of 34 min were collected. Measurements were performed in duplicates.

2D-HSQC NMR

Approximately 100 mg of sample was acetylated for better solubility 45 and then the residue was dissolved in 700 µL of DMSO-d6. The 2D HSQC NMR spectrum was acquired using the Bruker pulse program ‘hsqcetgpsi’ a relaxation delay of 1.7 s, a coupling constant of 145 Hz, an INEPT transfer delay time of 1.72 ms (d4 = 1/4J), 240 scans per increment , a spectral window of 10.5 ppm in F2 and 166 ppm in F1 with 1024 × 512 increments, and a spectral center set at 90.0 ppm in F1 and 5.3 ppm in F2. Central DMSO

(δC/δH=39.5/2.5 ppm) was used as an internal reference according to the solvent adopted.

————— Materials and Methods —————

13C-NMR

The 13C-NMR analysis were performed with the Bruker pulse program ‘zgig’ with 90C pulse width using an acquisition time of 1.4 s and a relaxation delay of 1.7 s. To provide complete relaxation of all nuclei, chromium (III) acetylacetonate (1.5 mg) was added inside the sample solution. A total of 24 000 scans were collected.

Dynamic Light Scattering (DLS) The particle size of the LignoBoost lignin and CleanFlowBlack lignin was measured using a dynamic light scattering meter (Zetasizer, Nano ZS, Malvern Instruments). For the measurements, solutions of lignin with a concentration of 1 gL-1 were used using 4:1 volume ratio of the dioxane/water mixture.

FTIR spectroscopy

IR spectra were collected using a Perkin-Elmer spectrophotometer (Spotlight 400 FTIR imaging system, Waltham, MA, USA) equipped with a Spectac MKII Golden Gate system (Creekstone Ridge, GA, USA). The samples were analyzed in the range of 600-4000 cm-1 with 16 scans at a 4 cm-1 resolution and a 1 cm-1 interval at the room temperature.

Thermal analysis

Thermal analysis was carried out on a TGA/DSC 1 (Mettler Toledo) instrument under the following operational conditions: heating rate of 10°C min-1, dynamic

25 ————— Materials and Methods —————

atmosphere of synthetic air or nitrogen (50 mLmin-1), a temperature range of 30-900°C, and sample mass of 2-10 mg.

Scanning Electron Microscopy (SEM) The structural characteristics of the samples were studied with a field-emission scanning electron microscope (FE-SEM, S-4800, Hitachi, Japan). All samples were coated with a 1 nm thick Pt-Pd layer sputtered with a Cressington 208HR high-resolution coater.

Cytotoxicity of lignin samples

Cell viability assay

MTT cell viability assay was used to measure formazan formation as an Table 1, Results from 2D NMR and C13 for different DHPs structure indicator of metabolic activity in cells. The cells were exposed to two types of technically produced lignin samples, including CFBL (Clean Flow Black lignin) and LBL (LignoBoost lignin) in the form of soluble lignin in media or encapsulated lignin in 6% laponite. The cytotoxicity tests were performed in two different sets of the experiment. In one set the cells viability was measured by MTT tests. The other experiment was life-dead assay which viability of cells was observed by fluorescence microscopy (see Paper VI for more details).

Method for bacterial reduction test

The antibacterial effect of the lignin samples was evaluated using a bacterial reduction test. Both gram-negative Escherichia coli ATCC 11775 (Sigma- Aldrich, Sweden) and gram-positive Staphylococcus epidermis (Sigma- Aldrich) were used for the bacterial reduction evaluation. Agitated nutrient

————— Materials and Methods —————

broth (Scharlab, Barcelona, Spain) was used for the cultivation of the bacteria. The cultivated bacteria were harvested through centrifugation, 5 000 rpm for 5 min, and washed twice by re-suspension in phosphate buffer, pH 7 (Sigma- Aldrich).

The bacterial reduction test was performed by incubating 0.15 g of lignin and lignin modified samples in a powder form in 10 mL phosphate buffer, pH 7, with a bacterial concentration of 106 CFU/mL, during 4 hours at 37°C. Regarding encapsulated lignin, 1 g of gel sample was used. The bacterial suspension was thereafter cultivated on Petrifilm aerobic count plates (3M, Sollentuna, Sweden) to determine the number of viable bacteria remaining after incubation with the different lignin samples (see Paper VI for more details).

27 ————— Results and Discussion —————

3. Results and Discussion 3-1 Effect of microenvironment on the synthesized lignin’s structure (Paper I) The well-known difficulties to separate biomass components in pure form regardless the chemicals and conditions used for various pulping processes led to the conclusion that covalent bonds may exist between lignin and carbohydrates forming so-called lignin-carbohydrate complexes (LCC).117 118 119 41 42 120 Also, some unexpected observations have been seen in many studies, i.e. there were differences between the structure of the softwood lignin, depending on whether it was covalently bound to arabinoxylan or glucomannan.120 It was suggested by Lawoko et al. 2005, that glucomannan- bound lignin seems to contain a higher number of condensed bonds and has a lower content of β–O-4 bonds, than the arabinoxylan-bound lignin.120 These differences can be correlated to the presence of the different types of monolignol in trees, meaning that softwood lignin consisting of phenolic units with a free C5 position has more chance to form the chemical linkages which will result in more branched structure than hardwood lignin.19, 121 To better understand some of these observations, many experiments were designed during this project to investigate the effect of microenvironment on the structures of lignin (Paper I). For this, DHP (dehydrogenase polymer lignin) was synthesized by using commercial lignin model compound at various pH values (3.5, 4.5, 6.5) aiming to mimic the presence of different hemicellulose in the tree during its growth. Based on the 2D HSQC NMR studies, we have shown that lignin which was synthesized at the lower pH, has more β-O-4 units

————— Results and Discussion—————

(generating to less condensed structures) than β-β plus β-5 units (generating more condensed structures). Shortly, there is higher non-condensed to condensed units value which means the formation of a more linear structure. This finding is in agreement with the previous claims by other scientists13, 120 that hardwood lignin has more linear structure than softwood lignin due to the presence of xylan with the acidic properties. In general, it is hard to distinguish and discuss the differences between the glucomannan- and arabinoxylan-bound lignins. In this respect, there have been studies showing that lignin can have covalent bonds to both glucomannan and arabinoxylan.120, 122 It seems that there might be a more direct effect of the hemicelluloses on the lignin structure. The carboxylic acid groups on the xylan might repel the local hydroxyl groups and attract more hydrogen ions which makes the xylan environment more acidic.

Scheme 1. A model explaining for the creation of non-condensed bonds at lower pH (reproduced-Paper I)

29 ————— Results and Discussion —————

As depicted in Scheme1, the path for the creation of both condensed structures demand more deprotonation which is the favor in less acidic conditions. As discussed, xylan’s microenvironment is more acidic (more H+), therefore, based on the suggested hypothesis less condensed structures, i.e., 5-5´ and β-5´ should favor. Whereas, the presence of both proton ions and water in a neutral environment leads to more β-O-4 structure in the lignin.123

Table 1. Measurement of the different amount of interunit structures by 2D NMR (reproduced-Paper I)

Finally, to understand the effect of microenvironment (different pH) on the lignin’s structure, as described, three different lignins were synthesized in three different pH conditions and products were analyzed by 2D HSQC NMR and 13C to quantify different units. According to these measurements (Table 1), the amount of non-condensed (β-O-4) to condensed structures (β-β & β-5) is quite high at low pH and reproducible in two different batches supporting the mentioned statement (Scheme 1).

————— Results and Discussion—————

3-2 Fractionation by ultrafiltration (Paper II)

As the aim of this PhD project has been a better understanding of the relationship between lignin structures, processing, properties and performance in various products. For this, the fractionation of technical (kraft) lignin by ultrafiltration was carried out at room tem, 3 bar transmembrane pressure (TMP) and 10% (w/w) sample concentration. The objectives were: a) to understand how the conditions (time) during the ultrafiltration process using ceramic membrane would affect the molecular weight of lignin fraction and b) evaluate physicochemical characteristics of the low molecular weight lignin (obtained by using membrane with molecular weight cut-off 1 kDa).

3-2-1 Impact of time on the membrane performance

The primary goal of the separation of LMw (<1000 Da) lignin was based on the knowledge of lignin being rich in phenolic content which gives special characteristics such as antioxidant44, 98 antibacterial124, 125 as well as an excellent unit for the further modification to tailor materials with new properties.126 A ceramic membrane with cut-off 1000 Da was used in this work to fractionate lignin (see Paper II). One of the objectives was to design a model system in a way to monitor the changes in the selectivity of the membrane with time during the cross-flow filtration of lignin solution (black liquor). Such changes may take place as a result of the fouling. Fouling127, 128, 129 is considered as one big problem for the membrane filtration which can affect the performance of the membrane over time. In parallel, specifically low Mw lignins were extracted for the further analysis of their antioxidant properties in the different active

31 ————— Results and Discussion —————

substances. Two sets of lignin samples were prepared by dissolving LignoBoost lignin (10% W/W) in the sodium hydroxide solution (5% W/W) for experiments including 1 LV-A, 1 LV-B, 3LRS and 4 LVWS (the total salt concentration for the mentioned experiments was 1.2 mol/L). For the other set of experiments with higher ionic strength, exactly the same concentration of LignoBoost lignin was prepared and a certain amount of NaCl was used to increase the ionic strength up to 2.4 mol/L. The experiments were performed using virgin membranes (no pre-treatment), re-used membranes (the membrane which one set of a trial run by it) and washed (Ultrasil 10 chemical) and stabilized membrane (with NaOH). The detailed description of each experiment are shown in Table 2. The total length of each ultrafiltration experiment was 100 h; however, liquid samples of permeate were withdrawn at the particular times, from time zero (the start time) up to 100 h, removing sample every 30 minutes. The experiment was run in a circulation mode for the whole sets.

Table 2. The description of sample preparation and experiment set up (reproduced-PaperII)

The permeate flux is reported based on the weight of liquid sample for the cross- flow ultrafiltration experiments of the two prepared LignoBoost Kraft lignin solutions, in higher and lower ionic strength (Fig.3).

————— Results and Discussion—————

Figure 3. Total liquid weight for the cross-flow ultrafiltration experiments with different samples and membrane treatment (taken-Paper II)

As shown in Fig.3, poor reproducibility between individual experiments were observed: 1LV-A vs 1LV-B and 2HR-A vs 2HR-B. The results were showing that the permeate fluxes were also unstable during 100 h operational time. These fluctuations were more evident for the experiments performed at higher ionic strength, particularly for experiments where re-used and cleaned with Ultrasil 10 membranes were applied (experiments 2HR-A and 2HR-B, see Table 2). Since experiments with higher ionic strength were performed with the reused and cleaned membranes (ultrasil 10 chemical), therefore, based on the implemented methods, it is hard to conclude that this flux incline is due to either the cleaning procedure with Ultrasil 10 and/or the increase in ionic strength. Moreover, to understand the effect of the washing step, 4 LVWS experiment was run at the virgin membrane which cleaned two times with Ultrasil 10 prior

33 ————— Results and Discussion —————

the trial and stabilized with 1 M NaOH. As obviously illustrated in Fig.3, the cleaning steps increase the permeate flux significantly which can indicate the point that how the chemical conditions and cleaning chemicals are affecting the performance of the membrane.

As illustrated in Fig.4 for three sets of experiments, there was a gradual shift in the Mw with increasing ultrafiltration time towards lower Mw values. This trend was more pronounced for virgin membranes (1LV-B), whereas, it gave more stable Mw values by increasing the number of cleaning cycles before the ultrafiltration experiment (4LVWS). It was found that the experiment with the twice-washed and the stabilized membrane, 4 LVWS, produced a lignin fraction of 600-500 Da over the entire 100 h of operation.

Despite these initial fluctuations for different experiments, all of them resulted in the lignin material with Mw of 400-500 Da after 100 h of ultrafiltration (Fig.4). Besides, no significant difference in average Mw of lignin was seen for the experiment performed at higher ionic strength. The lignin samples isolated by precipitation with sulphuric acid (6 M H2SO4) by placing inside the ice/water bath, from the final permeate fractions (100 H of ultrafiltration) were selected for the further structural analysis and property investigations, i.e., antioxidant activity.

————— Results and Discussion—————

Figure 4. Changes of Mw over the operation time (taken-Paper II)

3-2-2 Characterization of Low Mw lignin

The various functional groups of the low Mw lignin fraction and the initial LBL were quantified by 31P-NMR. As summarized in Table 3, the total phenolic OH- groups content and aliphatic group’s contents are higher in the initial LBL. However, the ratio of non-condensed to condensed phenolic fragments is higher in the low Mw fraction than in the initial LBL, suggesting a higher reactivity of this sample. The content of carboxylic groups is higher in the low Mw fraction indicating the high degree of oxidation of the lignin in this sample.

35 ————— Results and Discussion —————

Table 3. Measurement of different units by 31P-NMR (reproduced-Paper II)

Understanding the inter-unit structures can reveal and help more details about technical lignin and further modifications for material development. Moreover, the 2D HSQC NMR43, 14, 15, 130 was used to explain the different interunit linkages of initial LignoBoost kraft lignin and extracted low Mw lignin. The main purpose of the 2D NMR analysis was to observe the structural changes in lignin samples as a result of ultrafiltration through a 1 kDa membrane. For the original LignoBoost lignin, signals from the β-aryl ether (β-O-4), pinoresinol (β-β), phenylcoumarin (β-5), coniferyl alcohol structures and the methoxy groups can be clearly detected in the 2D NMR spectra (Fig 5a). Whereas these main structures had been disappeared for the extracted low Mw lignin, and mainly signals from coniferyl alcohol, methoxy groups from the phenolic part and free phenols predominantly appear (Figure 5b). This finding indicates the presence of smaller lignin fragments, likely monomers, dimers and oligomers, in the sample obtained as a result of fractionation by ultrafiltration. This finding is in accordance with the size of the sample (~400 Da) obtained by SEC.

————— Results and Discussion—————

Figure 5. Analysis of interunit linkages for a) original LBL and b) fractionated LMw sample (re-drawn-Paper II)

100 Original LBL

80 1 LV-B

60 2 HR-B

Percentage(%) 20

0 Total lignin Carbohydrates S-containing derivatives, % from C+L

Figure 6. The amount of lignin, carbohydrate and sulphur content in different fractions obtained by pyrolysis

37 ————— Results and Discussion —————

Fig.6 represents the major composition of different fractions of lignin. In both unfractionated (original LBL) and fractionated samples, the amount of lignin is very high, i.e. above 94%. Interestingly, the amount of degraded carbohydrate is around 2.7% before and after fractionation; however, sulphur content is higher in lignin samples after fractionation. This value was in good agreement with previously published work.47 The high content of phenolic groups in the polymer is often associated with the high antioxidant activity. However, as it was demonstrated previously98, the relationship is not so straightforward and substituting groups attached to aromatic lignin ring can influence lignin radical scavenging properties positively or negatively. For instance, as summarized in Table 6, the ratio (ArC1

+ ArC2) / (ArC3) for the low-Mw samples ( Mw~400-500 Da) are higher than that of the initial non-fractionated lignin, which enhances antioxidant activity.

Higher content of -OCH3 groups and phenols with CH2 in the Cα position (saturated chain) can increase the properties whereas the presence of double bond and oxygen in aliphatic side chain can have a negative effect. Based on the analytical pyrolysis (Table 6), the LMw lignins represent promising antioxidant properties with some substrates as discussed in the antioxidant section.

————— Results and Discussion—————

3-3 Chemical Modification of technical lignins (Paper III, IV, V, VI)

3-3-1 Physico-Chemical properties of technical lignins (LBL & CFBL)

LBL and CFBL, as technical lignins potentially available for the uses in materials applications, were characterized with different techniques, i.e., molecular weight (SEC), inter-unit structure (2D HSQC NMR), pyrolysis (Py- GC/Ms/FID), Thermal Analysis, cytotoxicity and antibacterial properties. Based on 2D NMR analysis results (Fig.7) there were no structural differences observed between these two technical lignins. Both LBL and CFBL contain all types of structural units which can be identified by 2D NMR method. Carbone number 2 of the phenolic unit was used as an internal standard to semi-quantify different structures. Corresponding to this quantification, the amount of total non-condensed structures for CFB lignin (lower Mw) was 21% and for kraft lignin 16% which is in good agreement with the result of 31P NMR (see Table 4). The condensed linkages are not detectable by 2D NMR analysis.

39 ————— Results and Discussion —————

Figure 7. Illustration of different interunit linkages in the original and fractionated lignins (LBL& CFBL) - (re-drawn-Paper III)

The molecular weights of LBL and CFBL, measured by the SEC/DMSO method, were approximately 5600 and 3000 Da, respectively (Table 4). As illustrated by the molecular weight distribution curves (Fig 8), there is one major peak for both samples; however, there is a shift toward the higher Mw

————— Results and Discussion—————

for LBL which is entirely in consistency with the obtained numbers. The main significant advantage of the fractionation of lignin is obtaining a more homogenous structure with low polydispersity (PD).

Figure 8. Analysis of molecular weight by SEC/DMSO (re-drawn-Paper III) 31PNMR60 as one technique was used to quantify the amount of various functional groups in different units in both lignin samples. Table 4 shows the total content of functional phenolic and hydroxyl groups in both lignin samples. CFBL had a higher content of phenolic and aliphatic hydroxyl groups than LBL (Table 4). In the measurement done by 31PNMR the amount of non-condensed to condensed units are higher for CFBL (having lower Mw). This observation was consistent with our previous work (Paper II) in which very low Mw lignin was extracted as a rich phenolic component possessing a higher number of non- condensed/condensed units and approved by 31PNMR.

41 ————— Results and Discussion —————

Table 4. Properties of LBL and CFBL measured by SEC/DMSO and 31PNM (reproduced-paper III)

Thermal properties

The thermal behavior of lignin was measured by TGA method and is illustrated in Fig.9. The destruction of the lignin macromolecule starts at 290°C for LBL and at 250°C for CFBL. It was found that the maximum destruction temperature

(Tmax) is similar for LBL and CFBL (414–416°C). However, the Tmax of polymer destruction differs between LBL and CFBL and had values of 390°C and 345°C, respectively.

Figure 9. Thermogravimetry (TGA) behavior of LBL and CFBL(Paper III)

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3-3-2 Amination of Technical Lignins (Paper III, IV, VI)

In addition to the fractionation, the various types of chemical modifications can be applied to the lignin to improve its reactivity in the synthesis of multiple products.

The purpose of the chemical modifications is to introduce reactive groups into lignin macromolecule. This can be done either by creating a new reactive site in the lignin aromatic ring or through the phenolic or aliphatic hydroxyl groups. In this project, the introducing the amine group via Mannich reaction was performed by using two different agents, 3-Aminopropyltriethoxysilane (APTES) and dimethylamine aqueous solution (Paper III and VI respectively). Furthermore, an active methacryloyl group was introduced through methacrylation reaction (Paper V) of hydroxyl groups. The purposes of these reactions were to improve the reactivity of lignin to synthesise the Lignin-Si hybrid materials, in the Lignin-St-DVB porous materials and to improve its antibacterial effect.

Mannich reaction by 3-Aminopropyltriethoxysilane (APTES) (Paper III, IV) Amination of lignin was done by 3-Aminopropyltriethoxysilane (APTES) using formaldehyde (Mannich reaction) to introduce amine group to increase the functionality of lignin for the creation of hybrid network through a sol-gel method in the next step. As can be seen in Scheme 2, formaldehyde makes a bridge at C5 to lignin moieties to the amine group.

43 ————— Results and Discussion —————

Scheme 2. Amination of kraft lignin with APTES. In the next step this produced product from this step was used for the creation of the silica network through a sol-gel reaction. FTIR confirmed the introduction of new substitute group (Fig.12, see section 3-4-1 for the further study).

Mannich reaction by Dimethylamine (Paper VI)

There have been many studies that how the presence of amine moieties can increase the antibacterial efficiency of the product. Since there are a lot of functional groups in lignin which can be modified, therefore, amination through Mannich reaction131, 132, 133 was selected as established secure method to produce this up-grade product. The Mannich reaction occurs as a result of the addition of dialkylaminomethyl groups in the presence of formaldehyde. As discussed by other scientists, based on the acidic or alkaline environment, they can produce N,N-dialkyl(methylene)ammonium ion(I) and N,N- dialkylaminomethanol (II) as an electrophilic species. In the lignin structure, C- 5 at the phenolic guaiacyl unit is very electron rich (nucleophile) and thus specific amino-alkyl group can sit in this position. Both acidic or alkaline conditions will result the same product.131, 132 This type of reaction under acidic

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condition implemented in this work to introduce amine group to the lignin backbone to further investigate the antibacterial properties.

Scheme 3. Mannich reaction mechanism between lignin and amines under acidic or alkaline conditions ( taken from Du et al. 133)

Figure 10. 13C-NMR for analysis of the Mannich reaction (Paper VI)

13 Since the modified lignin was soluble in d6-DMSO, the C NMR analysis was used to monitor the reaction progress and efficiency of the creation of new bonds. As clearly can be seen from the NMR spectra (Fig. 10), the Mannich reaction proceeded selectively and efficiently as the strong signal from aromatic

45 ————— Results and Discussion —————

C-5 (~115 ppm) disappeared and considerably shifted to downfield approximately at 122 ppm. Furthermore, signals belonging to the other atom remained intact, which confirms the selectivity of this reaction. Moreover, no noticeable side reaction could be observed, because not so many noisy peaks could be found. Regarding the substitution of an amine group, although it was not detected directly by either 13C nor H NMR, the carbon from methylene group adjacent to the amine group gave new peak approximately at 60 ppm and carbon from newly formed –N(CH3)2 around 44 ppm. These results are in a full agreement with the previous study as well.133

3-3-3 Methacrylation of lignin (Paper V)

Porous styrene-divinylbenzene (St-DVB) has been one the first-developed sorbents for the removal of inorganic as well as organic particles.134 The interaction between the analytes and the sorbents usually takes place through electron of the aromatic ring and/or Van der Waals forces.135 Therefore, the implementation of an organic substance with different functional groups can enhance the sorption properties significantly. For this reason, lignin with many different active functional groups on aromatic and aliphatic units can be incorporated in the network structure. There have been some works106, 87 where lignin was used in a mixture with St and DVB to produce porous materials. In spite of having porosity around 105-196 m2g-1, however, they did not perform promising adsorption. Therefore, this led to the point that to incorporate more amount of lignin without having an adverse

————— Results and Discussion—————

effect on the adsorption capacity. Besides, lignin activation with methacryloyl chloride gives more active sides to the St-DVB network. Methacrylation of lignin was confirmed by 13C NMR as described elsewhere.136 13C NMR (Fig. 11) confirms the appearance of the new peaks which corresponds to the presence of the methacryloyl group. Signals from –

CH3 group at 18 ppm, the –C=O signal at 166 ppm as well as double bond signals at 128 and 135 ppm. In parallel to 13C NMR to confirm the synthesis part, FTIR was utilized as the second support. The appearance of distinct signals associating to the carbonyl group (1728-1732cm-1) and the reduction at the phenolic hydroxyl group signals (3243 cm-1) which occurred for the modified lignin indicate the reaction creation (Paper V).

Figure 11. Analysis of the new substitution groups after methacrylation reaction by 13C-NMR (Paper V)

47 ————— Results and Discussion —————

3-4 Lignin materials (Paper III, IV, V, VI)

3-4-1 Lignin-Si hybrid materials (Paper III, IV)

The sol-gel method has been investigated and proven by many others scientists as a useful method for the generation of polymer-silica hybrid materials. In this method, the surface of the silica is modified by the phenol-containing compounds (lignin), which were previously activated through the Mannich reaction.137, 138 In this work, aminated lignin is incorporated in the silica network through a sol-gel reaction. Due to the presence of protons in the lignin structure, methylene bridge can be formed between lignin and APTES through the help of formaldehyde. Studied by other scientists133 suggesting that there is a Mannich condensation product in which nitrogen-containing groups can sit at the C5 position of the benzene ring, although, some different side reactions could happen. In the further step the functionalized lignin with nitrogen containing units, cohydrolyzed with TEOS in an acidic medium to form a silica network. To prove the creation of the network between lignin and silica, FTIR and XPS were utilized (Paper III).

Scheme 3. Reaction path for the synthesis of lignin-silica sorbent

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FTIR Analysis

Since lignin-silica hybrid compounds are not soluble in any solvent, therefore, NMR studies could not proceed for the confirmation. FTIR is one of the technique which can be used to monitor the bond creation in silica network. There is an intense adsorption band at 3412 cm-1 corresponds to the O-H stretching vibration in the hydroxyl group attached to the carbon atom. Clearly, the intensity of this band has been disappeared after the reaction, mainly due to some internal hydrogen bonds or interference with the silica network. The other important observation is the appearance of Si-O peaks. Two peaks at 1068 and 950 cm-1 correspond to the Si-O stretching vibration in addition to the peak at 795 cm-1 representing the deformational vibrations. All these peaks confirm the creation of the silica network (Fig.12)

Figure 12. Confirmation of silica network formation by FTIR (re-drawn-Paper III &IV)

49 ————— Results and Discussion —————

SEM analysis

SEM characterized the morphology of the original lignin and lignin-silica hybrid materials. As illustrated in Fig 13,14 for the original LBL and CFBL lignins, structures of the original lignins (Figure 13a-c,14a-c) are rather compact, whereas, after modification ( Figure 13d-f,14d-f) the morphologies of the lignin–silica hybrid composites become rougher, brittle and fragile which contains new forms of canals, cavities and irregular structures on the surface. The creation of these irregular porous like structures are somewhat atypical morphology for hybrid materials obtained by the sol-gel method. Therefore, this observation presumably confirms the creation of a silica network between the particles of lignin. Also, the porous features can enhance the effective surface areas of hybrid materials which can increase the capacity of the adsorption. LBL

Figure 13. The morphology of original LBL (a-c) and after lignin-silica network creation (d-f) (taken-Paper III)

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CFBL

Figure 14. The morphology of original CFBL (a-c) and after lignin-silica network creation(d-f)

(taken-Paper III) Silica Network

Figure 15. The morphology of synthesized silica (a-c)-(taken-Paper III)

Porosity measurement

The surface areas of the hybrid materials were analyzed by adsorption/desorption test. As summarized in table 5, the specific surface area increases substantially for hybrid materials, indicating the creation of a porous network. For example, the specific surface area has increased from 17.8 m2g-1 for LignoBoost lignin up to 74 m2g-1 for the LBL-silica hybrid as well as from 3.1 m2g-1 for original clean flow black lignin up to 92 m2g-1 for the CFBL-Silica

51 ————— Results and Discussion —————

hybrid. The increased capacity in hybrid material emphasizes their potential for adsorption applications. More importantly, by incorporation of silica in the lignin network, new lignin-based materials but with higher adsorption capacity was produced.

Table 5. Textural characterization of original and hybrid sample by N2 adsorption/desorption test (reproduced-Paper IV)

LBL-silica LBL CFBL-silica CFBL Silica

2 -1 SBET, m g 74.0 17.8 92.0 3.1 195.4

2 -1 Spores, m g 75.0 20.9 100.9 3.2 44.3

3 -1 Vpores, cm g 0.435 0.102 0.587 0.016 0.022

Dpores, nm 23.1 19.5 23.2 19.7 20.4

Thermal analysis

Thermal gravimetric analysis (TGA) was implemented to study the thermal properties of the original lignins and synthesized hybrids. The destruction processes were done at N2 atmosphere, and the mass loss is drawn versus the temperature to monitor the degradation pattern. As clearly can be seen from the TGA graphs, there is a similar degradation pattern for the corresponded samples. However, the difference between initial macromolecules and hybrids are apparent. In general, at the first stage of the decomposition, there is gradual moisture evaporation which occurs up to 150-200ºC. As illustrated in Fig.16, there is a gradual decomposition happening for hybrid materials which could indicate the movement of molecule segments inside the network before starting

————— Results and Discussion—————

the complete decomposition. The destruction of the lignin macromolecule begins at 290°C for LBL and at 250°C for CFBL. Moreover, maximum decomposition temperature (Tmax) was rather narrow for hybrid materials (414–

416°C). However, the Tmax of polymer destruction differs between LBL and CFBL and have values of 390°C and 345°C, respectively.

100 100

Figure 14. Thermal analysis of original and hybrid materials 80 80 CFBL-silica LBL-silica 60 60

Weight loss (%) Weight loss (%) CFBL 40 LBL 40

20 20 0 200 400 600 800 0 200 400 600 800 Temperature (oC) Temperature (oC)

0 0

-1 LBL-silica -1 CFBL-silica o T =414oC Tmax=416 C max

-2 -2 DTG (%/min) DTG

DTG (%/min) -3 -3 o Tmax=345 C LBL CFBL o Tmax=390 C -4 -4 0 200 400 600 800 0 200 400 600 800 o Temperature (oC) Temperature ( C)

Figure 16. Decomposition pattern of crude and constructed hybrid materials (Taken-Paper III)

53 ————— Results and Discussion —————

3-4-2 Lignin-St-DVB Porous microsphere (Paper V)

As discussed earlier, sorbents can be a promising candidate which can be used in different areas, including the adsorption of metals, organic and organic- inorganic contaminants.139, 106, 87 Depending on the application, sorbents can be prepared in different ways, however, some properties including a decent surface area, appropriate pore size and morphology are important parameters.135

Scheme 4. Different methacrylation steps, end up with the same product

Lignin-based porous microspheres were developed and characterized in this work to investigate the performance of the lignin as an eco-friendly substitution instead of styrene. Different types of lignin in non-modified and modified versions were used for the sample preparations. According to this study, it was shown that by the modification of low Mw lignin more uniform spheres with a surface area around 236 m2g-1 can be produced which is quite competitive with a commercially available St-DVB sorbent having a surface area of around 269 m2g-1. However, unmodified kraft lignin in combination with DVB confirmed the very promising surface area of about 409 m2g-1 (Paper V). As it is depicted

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in Scheme 4, the modification can be taken place by using 2 different moieties (path I and II); however, both paths result in the same final product. Path I was implemented for the modification of CFBL in this work. Thermal properties of porous microsphere The degradation behaviour of lignin-containing copolymers (lignin-St-DVB) was measured by thermogravimetric analysis (TGA). As it is depicted in Fig 17, almost for all the microspheres, the degradation happens between 400- 460ºC. For both two sets of samples, modified lignin and modified low molecular weight lignin’s, by increasing the amount of lignin the initial and maximum degradation temperatures decrease. However, in general, in the produced samples, the addition of lignin leads to the increased decomposition temperature and the formation of a higher amount of char. Furthermore, by incorporating styrene, the degradation temperature for both modified and unmodified lignins decreases. However, this decline shifts slightly towards the higher temperature by the addition of lignin, merely meaning that St-DVB copolymer represents the lower degradation temperature.

Figure 17. Comparison of different degradation pattern for various samples obtained by TGA technique (re-drawn-Paper V)

55 ————— Results and Discussion —————

Morphology To use microsphere for the adsorption purpose, sorbent particles should possess a uniform spherical shapes. 106, 87 Lignin as one eco-friendly material rather than styrene could be an alternative instead of styrene in the St-DVB copolymer. In respect to this purpose, several lignin-St-DVB copolymers were synthesized. Microspheres were prepared with two types of lignin, technical LBL and technical fractionated CFBL in crude and modified versions. SEM analysed the morphology of the microspheres. As illustrated in Fig 18, preferably all the samples contain uniformly distributed spheres with the size in a range of micrometres. First, as a general comparison between LMw lignin and industrial kraft lignin, microspheres formed by LMw lignin are more uniform. Secondly, as it is seen in Fig (18c-f and 18j-l), the spheres formed of LMw lignin-DVB give decent spherical shapes; however, they represent even more uniformity with the modified LMw lignin. Additionally, the amount of embedded lignin plays the role, i.e. there has not been any sphere formation in a low amount of kraft lignin and DVB mixture (Fig 18d-e). The addition of styrene improves the creation of spheres, more specifically, this is more pronounced for the industrial kraft lignin. As a conclusion, modified LMw lignin in a mixture with DVB, forms better microspheres even in the absence of styrene which bring the new great possibility for lignin as a sustainable and environmentally friendly substance to be substituted by styrene.

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Figure 18. Morphology studies of sphere particles by SEM (taken-Paper V)

3-4-3 Lignin nanoparticles (Paper VI)

In this work (Paper VI), lignin nanoparticles were prepared with the purpose of analysing their potential for the antibacterial application. Revolving lignin with an irregular structure to sphere shapes with a micro and nano dimensions can positively improve lignins properties.140 Lignin sphere micro-nano particles in this project were prepared according to the recently developed method.114

57 ————— Results and Discussion —————

Corresponding to this study114, type of solvent used to dissolve lignin can have a significant impact on the particle shapes and sizes. It was revealed by Lievonen et al114 that lignin nanoparticles prepared from THF are more spherical and uniform rather than from the ethylene glycol dissolved lignin. It was discussed that it could be due to the better solubility of lignin in THF and consequently suitable solvent exchange interaction with water. Corresponding to this study, the pre-dialysis concentration of lignin solution affects the particle size. They showed that pre-concentration between 1-10 mg ml-1 resulted approximately in the same particle sizes; therefore, this pre-dialysis concentration was used in this work for the nanoparticle preparation, for both LBL and CFB lignins. The primary purpose of this production was to make very regular spherical particles in a range of nanometer. The morphology of the produced particles is depicted in Fig.19. It can clearly be seen that, while the original lignin samples had very heterogeneous and irregular structure, the corresponding nanoparticles, on the other hand, have regular spherical shapes. Despite the regular shapes, the dimensions of the particles vary between 1.5 µm to a couple of nanometers, which indicates not very well distributions of the particles size. In spite of exact repetition of the same method, this particle size polydispersity was not observed by the other scientists.114

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a. Original LBL b. LBL-sphere nanoparticles

a. Original CFBL b. CFBL-sphere nanoparticles

Figure 19. Morphology studies of lignin sphere micro-nano particles (Paper VI)

3-4-4 Lignin hydrogels (Paper VI)

Silicate nanoplatelets (laponite~LP) was proved and utilized for the hydrogel preparation regarding biomedical applications141, 142. The main character of utilizing laponite is due to it biodegradability inside the body. Laponite based shear-thinning hydrogels have a special place for the many types of biomedical applications, e.g. chemoembolization.141 Injectability of these materials through catheter could make it more applicable for the different medical treatments. The surface of silicate nanoplatelets has positive and negative charges. Therefore, many different chemical units can be embedded inside this highly charged material to create new bonds through electrostatic forces, which can bring new properties to the hydrogel. Due to the antibacterial properties of lignin,124 the preparation of injectable lignin hydrogel with the aim of an antibacterial application was considered in this work (Paper VI).

59 ————— Results and Discussion —————

Lignin and silicate nanoplatelets were mixed with the different ratios of lignin and silicate nanoplatelets. The purpose was to increase the lignin amount inside the hydrogel network to obtain the desired properties and simultaneously to control the injectability of the blend. The instron machine measured the injectability of samples. One of the observation was that in some ratios, e.g., by decreasing lignin content there was a phase separation between the two components. Also, by increasing the lignin amount the final hydrogel lost its injectability. As it is summarized in Fig.20a.b, the mentioned ratios gave better injectability and phase mixture. Between the ranges of these ratios, the hydrogels are injectable through the catheter. The other observation is that for both lignins, within the mentioned ratios, still by increasing the lignin ratio the final hydrogel becomes stronger and less flowable as the injection force (N) goes up from approximately 10 N to 15-20 N. Therefore, according to the application the behaviour of these gels can be tuned.

Figure 20. Measurement of the injectability of hydrogels by Instron for a) LBL and b) CFBL based hydrogels (Paper VI)

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3-5 Application of lignin materials

3-5-1 Antioxidants (Paper II)

It has been reported earlier, that lignin as a polyphenolic compound, possess good antioxidant properties.143, 144, 44 In this regard, to get more objective results, the antioxidant activity of the samples under this study including original LBL and fractionated low Mw lignins were characterized by using different methods. It is very crucial to point out that parameters like the mechanisms of the reactions with free radicals, kinetic parameters and other factors are determining at the antioxidant activity of the substance as described elsewhere.143, 144 As demonstrated previously98, 145, certain structural features attaching to lignin benzene ring can influence lignin radical scavenging properties in positive or negative ways. For instance, as summarized in Table

6, the ratio (ArC1 + ArC2) / (ArC3) for the low-Mw samples ( Mw~400-500 Da) are higher than that of the initial non-fractionated lignin, which enhances antioxidant activity. More content of guaiacyl (G) phenols (OCH3 groups) are higher, and phenols with CH2 in the Cα position can increase the properties whereas the presence of double bond and oxygen in aliphatic side chain can have a negative effect. Therefore, these types of LMw lignins represent very promising antioxidant activities with some substrates as discussed in the antioxidant section.

61 ————— Results and Discussion —————

Table 6. Table 6. Summary of certain structural features attaching to lignin benzene ring- measured by pyrolysis test (reproduced –Paper II)

As illustrated in Table 7, the results of the ABTS●+ and DPPH tests are -1 interpreted regarding the IC50 (mg L ). IC50 is defined as the concentration of the sample required for the 50% inhibition of the radical species. Therefore, the lower amount is considered as effectiveness of the sample. For ABTS●+ scavenging, the low-Mw fraction exhibited better antioxidant activity than the unfractionated LignoBoost lignin. However, neither initial nor fractionated lignins did not give promising radical scavenging properties in a test with DPPH. The results of the ORAC assay (Table 7) conform to those observed in the ABTS+ test. Moreover, low Mw lignin represents three times higher antioxidant activity than the commercial antioxidant Trolox. Such a

————— Results and Discussion—————

discrepancy between lignin sample efficiency in the DPPH test, and the two other assays can be explained by the difference in the mechanisms of the radical scavenging reactions in the tests applied.143

The low-Mw fraction, which demonstrated high results in the ABTS●+ and ORAC tests, can be considered as a great candidate as a radical scavenger in lipid-rich products. More specially, foods containing the significant amount of polyunsaturated fatty acids which can easily be decomposed, need radical scavenging substance to stay longer. According to European regulations (EC 2004), due to the many health-relating concerns, the concentration of synthetic antioxidants in foods should be kept below 0.2 mg g-1 of the oil or fat content in such foods. However, there are no strict regulations concerning the naturally derived antioxidants, which explains the growing interest in the use of these type of compounds.146 As is shown in Table 7, generally, low Mw lignin demonstrated higher antioxidant stability than LignoBoost lignin. However, the low Mw fraction was an efficient antioxidant even at relatively low concentration. In comparison with a commercially added antioxidant, TBHQ, the addition of 5 mgg-1 of low Mw lignin can compensate the same stability effect of the addition of 1 mgg-1 TBHQ. On the other hand, unfractionated lignin did not show such a promising effect, though; with increasing the amount the performance gets better.

63 ————— Results and Discussion —————

Table 7. Summary of antioxidant activity of lignin samples in different substrates (taken, Paper II)

Radicals Lignin samples

LignoBoost lignin Low-Mw fraction

DPPH●, IC (mg L-1) 23.7 ± 0.9 32.8 ± 1.0 50 ABTS●+, IC (mg L-1) 5.5 ± 0.1 3.9 ± 0.2 50

a ORAC assay (TE mmol g-1) 9.09 ± 0.1 14.9 ± 0.2 Inhibitory properties of lignin samples in mayonnaise oxidation (Oxipres test)

Additive concentration, mg Induction period (IP), h g-1 0 1.9 1.9 2.5b 1.93 2.27 5.0 2.73 3.39 Protection factor 0 1 1 2.5b 1.02 1.19 5.0 1.44 1.78 a Antioxidant capacity calculated based on Trolox; 1 g of Trolox corresponds to 3.9 TE mml g-1 b Addition of 1 mgg-1 (0.1%) TBHQ to a mayonnaise sample resulted in an IP of 3.48 h and a protection factor of 1.83

3-5-2 Sorbents (Paper III, IV)

Lignin-silica hybrid materials were synthesized with the purpose of the development of porous materials for the adsorption of organic pollutants, e.g.

————— Results and Discussion—————

dyes from the textile industry sewage. Since lignin has different functional groups on its backbone, more specifically negative groups, therefore it can be a great candidate for the adsorption of positively charged substances. In general, hybrid materials have very rough and irregular structures which could interrupt the adsorption efficiency to some extent. Therefore, understanding the adsorption rate is important to evaluate the efficiency of the hybrid materials, on a large industrial scale. In this respect, the kinetic test was performed to understand how the contact time between two phases and concentration would affect the adsorption rate. For this purpose, methylene blue dye was prepared in three different concentrations (10, 30 and 50 mgL-1) and was adsorbed by the prepared hybrid sorbents in 48 hours’ period. As shown in Fig.21a the adsorption takes place in two regions. A rapid adsorption occurs during the first 3-4 hours and after it inclines slowly. One main indication could be that at the initial step, due to the presence of numerous vacant porous places, there is rapid adsorption, and consequently more adsorption between adsorbate molecules and the vacant adsorption sides of the adsorbent surface. However, after the majority of adsorption sides are occupied, later on, there is very slow adsorption. Fig. 21a represents that the equilibrium was established after 4 and 8 hours for CFBL and LBL samples respectively. This indicates the better affinity toward CFBL hybrid which is in correlation with the promising porosity capacity which was higher for CFBL hybrid rather than LBL hybrid. It should be mentioned that the highest adsorption capacity was reached after 48 hours interaction time; however, the nearly 50% and 80% of adsorption occurred at the first 30 minutes for LBL and CFBL hybrids respectively.

65 ————— Results and Discussion —————

a b

Silica 70 LBL LBL-Silica 60 58,9 CFBL CFBL-Silica 50 41,6 40 31,2 32,3 30 20

Adsorption capacity,mg/g 10 3,9 3,9 0

Figure 21. Comparison of the adsorption kinetics for the different hybrids (reproduced & taken- Paper III &IV)

Fig. 21b compares the adsorption capacity between un-modified and modified lignin-based products. The apparent discrepancy on adsorption capacity was observed by shifting from original un-modified lignins toward the hybrid compounds. It was clearly shown that the hybrid materials consisting of a porous structure have more adsorption capacity. Moreover, CFBL-silica hybrid presents more adsorption capacity, mainly due to the presence of more functional groups and consequently the creation of better porous compounds with more adsorption sides.

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3-5-3 Antibacterial (Paper VI)

Cytotoxicity test (Cell viability of soluble lignin)

So far, there have been only a few studies147, 148 to evaluated cytotoxic effects of lignins; however, their potential applications in skin care products and pharmaceutical industries make it essential to assess their harmlessness. Herein, we investigated the cytotoxic effect of two compounds of lignin, i.e. CFBL and LBL in murine fibroblasts using MTT assay. The obtained results revealed concentration and time-dependent cytotoxic effect of both the compounds (Fig 22. A & B). LBL seems to be more cytotoxic as compared to CFBL, especially, after a longer exposure time like 48 and 72 h. It was found in this study that LBL, having a higher molecular weight, represents more toxicity in comparison to the fractionated CFBL. For two sample, by shifting from 24 h to 72 h, the toxicity rises with the increased exposure time of lignin samples with the cells. However, this observation is more pronounced for the LBL lignin. The other crucial parameter is a concentration of the initial sample. It was observed that by increasing the concentration from 250 µg to 500-1000 µg, the toxicity goes up substantially. Once more, this is quite notable in the case of LBL. These observations are in a high agreement with dead-life experiments (Fig.22 C-D). The proliferation of cells goes down by increasing the initial sample concentration in both samples. However, this decline is not that sharp and still, a decent number of viable cells was seen. The evaluation of the dead-life test is based on the dye adsorption capability, merely meaning that all the live cells

67 ————— Results and Discussion —————

uptake green colour and dead cells the red colour. It is evident that in a comparison between green and red colours, the dominant one is the green colour, which indicates the proliferation of live cells.

Furthermore, in respect to the presence of dead cells, it cannot be concluded that this was the result of the lignin toxicity, mainly because these type of red colours were found in the reference sample as well. Corresponding to our studies, LBL shows more toxicity than CFBL, especially in higher concentrations. And this could be due to the high Mw as well as higher polydispersity of LBL rather than CFBL. However, this is in contradictory with the other scientist’s studies in which they concluded that higher Mw lignin represented lower toxicity.124 This opposite finding could be due to the different type of lignin and the extraction methods which are affecting lignins’ properties.

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A:CFBL B:LBL

Figure 22. A) Effect of different concentrations of CFB on fibroblast cell viability at different incubation times. B) Effect of different concentrations of LBL on fibroblast cell viability at different incubation times. C) Representative live/dead images of fibroblast cells after incubation with different concentrations of CFBL for 24 and 72 h. D) Representative live/dead images of fibroblast cells after incubation with different concentrations of LBL for 24 and 72 h. (Paper VI)

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Beside, the cytotoxicity measurement was done for the encapsulated lignin as well. The results are illustrated in Fig.23. The main aim of this test was to analyze the toxicity of hydrogel in murine fibroblasts using MTT assay. Corresponding to the live-dead results cell viability is decent in the present of the hydrogel as well. Cells grew in the presence of hydrogel. Therefore, this type of hydrogel can be considered for the further evaluation and development for the biomedical applications. Fig.23 represents the summary of MTT test for the viability as well. In comparison between the reference and samples, not a significant deduction can be seen, meaning no toxicity for the gels as well. There is a small deduction down to 89% for a higher concentration of encapsulated LBL lignin.

————— Results and Discussion—————

Figure 23. A) Representative live/dead images of fibroblast cells after incubation with encapsulated CFB in laponite (LP) for 24 and 72 h. B) Representative live/dead images of fibroblast cells after incubation with encapsulated LBL in laponite (LP) for 24 and 72 h. C) Representative live/dead images of fibroblast cells after incubation with 6% laponite (LP) gel for 24 and 72 h. D) Cytotoxicity of encapsulated lignins.(Paper VI)

Antibacterial Properties As discussed before, the main purpose of development of chemically modified lignins, including preparation of lignin nanoparticles and aminated lignins and incorporating of these materials for the development of lignin-based injectable hydrogel were to investigate their potentials against bacteria. Two bacteria lines, gram-positive (S. epidermis) and gram-negative (E. coli) were examined for these trails. Our observation was in front with other scientist’s who found 124 that crude lignins in a powder form represent antibacterial activity against gram-positive, however, it can depend on the lignin’s source and the implemented extraction methods. Lignin activity against bacteria mainly concerns gram-positive.124 In this work, the antibacterial activity of unmodified lignins (crude LBL and CFBL) plus modified lignins (nanoparticles and aminated samples) were examined against two above mentioned bacteria.

71 ————— Results and Discussion —————

As obviously can be seen in Fig. 24, in the test with gram-positive (S. epidermis), all the crude samples in unmodified and modified versions can inhibit bacteria’s growth. Whereas, in the test with gram-negative (E. coli), no promising inhibition was observed for the un-modified samples as well as the nanoparticles (Fig 25). However, as it is illustrated, aminated samples represent quite promising inhibition properties. This observation can be very promising for using lignin as an antibacterial substance with a simple amination substitution. Antibacterial activities were tested for the encapsulated lignins as well, though, not promising antibacterial property was observed for the encapsulated lignins except hydrogel which produced of NanoCFBL against gram-positive. In respect to this observation, one interpretation could be that nanoparticle can diffuse and lash out and get in contact with bacteria’s cell wall. However, this observation demands more experimental repetition and more detailed analysis. The other reason for the less-efficient properties of these gels could be that lignin is completely encapsulated inside the silicate nanoplatelets through electrostatic bonds and 4 hours incubation time was not sufficient for the encapsulated lignin to diffuse out and contact with the bacteria. For the better understanding and studying the efficiency of these developed hydrogels, more research with respect to the hydrogel network development and antibacterial studies are strongly demanded.

————— Results and Discussion—————

Test with S. epidermis, gram-positive

Figure 24. The antibacterial properties of lignin sample in test with gram-positive.(Paper VI)

Test with E. coli, gram-negative

Figure 25. The antibacterial properties of lignin sample in test with gram-negative.(Paper VI)

73 ————— Conclusions —————

4. Conclusions Technical lignin as one of the significant side product of the chemical pulp mill, demands lots of studies to pave the path for further modifications and valorisations. In parallel to the technical characterizations, some fundamental studies are required to reveal lignin’s structure in more details.

 By synthesis of lignin in a different microenvironment, i.e. different pH including pH 3.5, 4.5 and 6.5, to mimic the biological environment around the lignin, it can be seen that how lignin’s structure can get influenced in various microenvironments. Our observation was following the previous hypothesis that lignin bound to xylan has more linear structure than lignin bound to glucomannan. It was observed that the acidic microenvironment around xylan favours the creation of more β-O-4 structure (Paper I).  Fractionation of technical lignin to the more homogenous and low Mw materials is strongly suggested as a necessary step in the applications of lignin in various products. For instance, in the antioxidant studies with the un-fractionated and fractionated lignin, it was observed that low Mw lignin demonstrated better radical scavenging properties, although, depending on the different substrate, (Paper II).  The presence of active functional groups in lignin molecule give the personal potential for lignin applications in various polymeric materials. Lignin-silica hybrid materials, prepared from aminated lignin, were

————— Conclusions —————

developed and demonstrated very promising adsorption capacity for methylene blue dye. The results proved that hybrid composite formed by CFBL represented the higher surface area and porosity and consequently have better adsorption capacity, (Paper III, IV).  Furthermore, in a Lignin-St- DVB composites, synthesized from the non-modified and methacrylated lignins, it was found out that lignin can replace partly styrene component. It can be an exciting result since lignin comes from more green and sustainable source. The methacrylation of lignin improved its reactivity and resulted in polymeric porous microspheres with improved morphology and properties, (Paper V).

 The lignin-based injectable hydrogel was developed by the combination of lignin with silicate nanoplatelets. Un-modified crude lignins and also differently modified lignins, including lignin nanoparticles and aminated lignin were used. Crude samples of lignin, including unmodified LBL and CFBL lignins, and produced crude nanoparticles and aminated lignins represented promising inhibition in the test with gram-positive. In spite of this observation, no antibacterial property was found against gram negative except the aminated samples. This finding can be very optimistic about the future applications of lignin, i.e., for biomedical adhesives and cosmetics. However, encapsulated lignins did not show any good inhibition properties except nano-CFBL gel against gram positive. Therefore, to utilize these types of gels to any biomedical applications, more studies and analysis are demanded, (Paper VI).

75 ————— Future Outlook —————

5. Future Outlook Based on the studies through this thesis in respect to the technical lignin’s applications for the high value-added materials, for any further application, structural characterizations is strongly recommended. Since technical lignin suffers a lot with the heterogeneity, therefore, fractionation with different methods are suggested. In this respect fractionation with ceramic membrane which can be directly implemented in the industry due to its higher chemical and temperature tolerance, is one alternative. The best suggestion would be to separate lignin with ceramic membranes with different cut offs to improve homogeneity and decrease poly-disparity. Therefore, sequential fractionation of technical lignin with different cut-offs is suggested. After separation of lignin with different sizes, more detailed structural analysis are required to investigate the properties and reactivity of the different fractions.

Moreover, along this thesis it was found out that how introducing of new moieties to the lignin’s backbone can enhance its properties accordance with the final applications. For instance, lignin with having intrinsic antibacterial properties can have a great potential as an antibacterial species with having application in different ways. To obtain extra antibacterial properties, new moieties can be introduced for boosting the property. More studies against different bacteria strains are recommended for more detailed results.

Additionally, converting un-regular lignin to the regular nanoparticle can bring new type of property to this material which demands more studies.

To sum up, fractionation of technical lignin plus chemical modifications are strongly suggested for utilization of technical lignin for high value products.

————— Acknowledgements —————

6. Acknowledgements This work is a reflection of hard work put in by a number of people and I wish to appreciate all of them.

I would like to begin by expressing my sincere gratitude to Prof.Mikael.E.Lindström for giving the opportunity to pursue research, his constant support and help and enabling a thriving environment to fulfill this big part of my education. He is gratefully acknowledged for the patient supervision and inspiring discussions.

I had a pleasure of having two helpful co-supervisors. I would like to deeply appreciate and express my regards to Dr.Olena Sevastyanova and Prof.Gunnar Henriksson for all the scientific helps and great discussions along this thesis with the different projects.

Wallenberg Wood Science Center (WWSC) is gratefully acknowledged for funding of my PhD.

I would like to express my deepest acknowledge to Prof.Lars Berglund and my main supervisor, Prof.Mikael.E.Lindström of giving the chance for me to be a visiting scholar in US. This period of my PhD created new and unforgettable scientific views which will affect my future career positively.

I am so thankful for the current and mentor colleagues at FPT, more specifically, all the colleagues and friends from WWSC. I spent wonderful and unforgettable moments along WWSC schools and workshops with you all. Friendship established during this time have played an important role in mitigating the tense moments. We will stay friends. Jennie, Saina, Nicola, Pär, Dr.Alireza, Dr.Ramiro Rojas, Dr.Mikaela Helander, Dr.Linda Fogelström, Dr.Daniel Söderberg, Hanieh, Malin, Tahani, Marcus, Tobias I, Joakim, Tobias B, Pernilla, Nitesh, Lilian, Thomas, Justin, Dr.Martin Lawoko, Dr.Yuan Yuan, Dr.Qiliang, Dr.Qiong, Dr.Anastasia, Dr.Claudio, Dr.Francisco, Dr.Jakob Dr.Natalia, Dr.Diana, Dr.Peter, Dr.Giada, , Prof.Monica Ek, Anna Ottenhall,

————— Acknowledgements —————

Carl, Dr. Danila, Chao, and rest of friends, I would like to express my gratitude and best wishes for all of you in your private life and your career.

I deeply appreciate Prof.Mats Johansson for reviewing my thesis.

Furthermore, I would like to thank all the members of Project I and wood division members for the group meetings and collaborations. All the co-authors are appreciated for their works and collaborations.

I wish to take a chance and appreciate colleagues in US. I would like to express my gratitude and appreciation to Prof.Ali Khademhosseini and his members of giving the chance for joining to his group and collaboration. Tough but full of inspiring and concrete projects and educations.

Most importantly, I am forever grateful to my family, specially my parents, who have made numerous support and help to provide me with the best, and for exemplifying dedication, kindness and hard work. Their blessing, constant motivational support and love have been determined where I am standing now. You are standing far away, but always in my hard. Love you.

Stockholm, 2018 Selda Aminzadeh

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