Betulin-Modified Cellulosic Textile Fibers with Improved Water Repellency, Hydrophobicity and Antibacterial Properties

Betulin-modified cellulosic textile fibers with improved water repellency, hydrophobicity and antibacterial properties

TIANXIAO HUANG

Licentiate Thesis KTH Royal Institute of Technology School of Engineering Sciences in Chemistry, Biotechnology and Health Department of Fiber and Polymer Technology SE-100 44, Stockholm, Sweden

ISBN 978-91-7873-109-1 TRITA-CBH-FOU-2019:14 ISSN 1654-1081

Akademisk avhandling som med tillstånd av KTH i Stockholm framlägges till offentlig granskning för avläggande av teknisk licentiatexamen torsdagen den 28 Feb 2019 kl. 10.00 i Rånbyrummet, KTH, Teknikringen 56, Stockholm.

Abstract Textiles made from natural sources, such as cotton and flax, have advantages over those made of synthetic fibers in terms of sustainability. Unlike major synthetic fibers that have a negative impact on the environment due to poor biodegradability, cotton cellulose is a renewable material. Cotton cellulose fibers exhibit various attractive characteristics such as softness and inexpensiveness. Cellulosic textiles can be easily wetted, since the structure contains a large amount of hydrophilic hydroxyl groups, and when water repellency is needed, this is a disadvantage. Currently, paraffin waxes or fluorinated silanes are used to achieve hydrophobicity, but this contradicts the concept of green chemistry since these chemicals are not biodegradable. The use of bio-based materials like forest residues or side-streams from forest product industries might be a good alternative, since this not only decreases the pressure on the environment but can also increase the value of these renewable resources. Betulin is a hydrophobic extractive present in the outer bark of birch trees (Betula verrucosa). Nowadays, the birch bark containing betulin generated in the paper industry is disposed of by incineration as a solid fuel to provide energy, but this application is not highly valuable and this motivates us to see whether betulin can be used as a hydrophobe to prepare waterproof cellulosic textiles. Methods of dip-coating, film compression molding and grafting were performed to build “betulin-cellulosic textile system” to render the textile with hydrophobicity and other functions. The textile impregnated in a solution of betulin-based copolymer exhibited a contact angle of 151°, which indicated that superhydrophobicity can be reached. AATCC water spray test results showed that cellulosic textile coated with betulin-based film had a water repellency of 80, which is the third highest class according to the rating standards. Betulin-grafted textiles were also prepared and showed a static water contact angle of 136°, and an antibacterial property with a bacterial removal of 99%. This thesis proposes that betulin can be used as a green alternative in functional material preparation. By developing betulin, a more value-added application rather than incineration can be achieved.

Keywords Antibacterial, Betulin, Biorefinery, Cellulose, Grafting, Coating, Hydrophobicity, Textile, Water repellency

Sammanfattning Textilier gjorda av naturliga källor, såsom bomull och lin, har fördelar jämfört med de syntetiska fibrerna vad gäller hållbarhet. Till skillnad från stora syntetiska fibrer som har en negativ inverkan på miljön på grund av dålig biologisk nedbrytbarhet är bomullscellulosa ett förnyelsebart material. Bomullcellulosafibrer uppvisar olika attraktiva egenskaper, såsom mjukhet och kostnadseffektivitet. Cellulosa textiler kan lätt fuktas, eftersom strukturen innehåller en stor mängd hydrofila hydroxylgrupper, och när vattenavstötning behövs är detta en nackdel. För närvarande används paraffinvaxer eller fluorerade silaner för att uppnå hydrofobicitet, men detta strider mot konceptet grön kemi, eftersom dessa kemikalier inte är biologiskt nedbrytbara. Användningen av biobaserade material som skogsrester eller sidoströmmar från skogsindustrin kan vara ett bra alternativ, eftersom detta inte bara minskar trycket på miljön utan också kan öka värdet av dessa förnybara resurser. Betulin är ett hydrofobt extraktionsmedel i ytterbarken av björkträd (Betula verrucosa). Numera bortskaffas björkbarken som innehåller betulin i pappersindustrin genom förbränning som ett fast bränsle för att ge energi, men denna applikation är inte mycket värdefull och detta motiverar oss för att se om betulin kan användas som hydrofob för att förbereda vattentätt cellulosa textilier. Metoder för dipbeläggning, filmkompressionsgjutning och ympning utfördes för att bygga "betulin-cellulosahandelssystem" för att göra textilen med hydrofobicitet och andra funktioner. Textilen impregnerad i en lösning av betulinbaserad sampolymer uppvisade en kontaktvinkel av 151 °, vilket indikerade att superhydrofobicitet kan nås. Resultat av AATCC-vattensprutning visade att cellulosad textil belagd med betulinbaserad film hade en vattendämpning på 80, vilket är den tredje högsta klassen enligt betygsnormerna. Betulin-ympade textiler framställdes också och visade en statisk vattenkontaktvinkel av 136 ° och en antibakteriell egenskap med bakteriell borttagning av 99%. Denna avhandling föreslår att betulin kan användas som ett grönt alternativ vid framställning av funktionellt material. Genom att utveckla betulin kan en mer mervärdesansökan snarare än förbränning uppnås.

Nyckelord Antibakteriell, Betulin, Bioraffinaderi, Cellulosa, Ympning, Beläggning, Hydrofobicitet, Textil, Vattenavvisande

List of publications

Paper I Water repellency improvement of cellulosic textile fibers by betulin and a betulin- based copolymer Tianxiao Huang, Dongfang Li, Monica Ek. (2018) Cellulose 25:2115-2128 doi:10.1007/s10570-018-1695-5

Paper II Hydrophobic and antibacterial textile fibres prepared by covalently attaching betulin to cellulose Tianxiao Huang, Chao Chen, Dongfang Li, Monica Ek. (2019) Cellulose https://doi.org/10.1007/s10570-019-02265-8

Contributions of the author to these papers: Paper A Principal author of the manuscript. Planned and performed the experimental work. Paper B Principal author of the manuscript. Planned and performed most of the experimental work. The antibacterial assay and related text were carried out by Chao Chen.

Contributions to conferences: 5th ISETPP & 3rd IPEC, South China University of Technology, 7-9 November 2016, Guangzhou, China. A poster “Hydrophobic Coating on Cellulosic Textile Material” was presented and awarded a second prize.

Cellulose Materials Doctoral Students Conference 2017. 23-25 Oct 2017, Retzhof/Wagna, Austria. Oral presentation: “Hydrophobic Coating on Cellulosic Textile Material by Betulin and a Betulin Based Copolymer”

15th European Workshop on Lignocellulosics and Pulp (EWLP 2018). 26-29 June 2018 Aveiro, Portugual.

Poster “Hydrophobic cotton fabrics decorated with betulin moieties prepared through TEMPO-mediated oxidation and esterification”

5th International Conference on Pulping, Papermaking and Biotechnology (ICPPB’18). 12-14 November 2018, Nanjing, China. Oral presentation: “Fabrication of hydrophobic cotton textiles with antibacterial property by covalent introduction of betulin”.

Abbreviations

AATCC American Association of Textile Chemists and Colorists CC Carboxyl content DO Degree of oxidation DSC Differential scanning calorimetry E. coli Escherichia coli FTIR Fourier transform infrared spectroscopy NMR Nuclear magnetic resonance spectroscopy S. aureus Staphylococcus aureus SEC Size exclusion chromatography SEM Scanning electron microscopy TEMPO 2,2,6,6-tetramethyl-1-piperidinyloxy TMS Tetramethylsilane TPC Terephthaloyl chloride WCA Water contact angle

Contents 1 Introduction ...... 1 2 Background ...... 2 2.1 A brief introduction to wood chemistry ...... 2 2.1.1 Structure and property of cellulose ...... 2 2.1.2 Extractives from birch bark ...... 3 2.2 Textile hydrophobization ...... 3 2.2.1 A brief introduction to wetting theory ...... 3 2.2.2 Methods to obtain hydrophobic cellulose textile fibers ..... 5 2.3 Nucleophilic acyl substitution ...... 5 2.4 TEMPO-mediated oxidation ...... 6 2.5 Aim and objectives ...... 7 3. Experimental ...... 8 3.1 Materials ...... 8 3.2 Pretreatment of cellulosic textile ...... 8 3.3 Preparation of a water-repellent cellulosic textile by impregnation and compression molding (Paper I) ...... 8 3.4 Preparation of hydrophobic and antibacterial cellulosic textile by surface chemical modification (Paper II) ...... 10 3.5 Characterization ...... 10 3.5.1 Nuclear magnetic resonance (NMR) ...... 10 3.5.2 Fourier transform infrared spectroscopy (FTIR) ...... 11 3.5.3 Size exclusion chromatography (SEC) ...... 11 3.5.4 Differential scanning calorimetry (DSC) ...... 11 3.5.5 Scanning electron microscopy (SEM) ...... 11 3.5.6 Energy dispersive X-ray analysis (EDS) ...... 11 3.5.7 Static water contact angle (SWCA) measurement ...... 11 3.5.8 AATCC water repellency test ...... 11 3.5.9 Carboxyl content (CC) determination ...... 12 3.5.10 Degree of oxidation (DO) ...... 12 3.5.11 Tensile test ...... 12 3.5.12 Antibacterial assays ...... 12 4 Results and Discussion ...... 14 4.1 Impregnation and compression molding to prepare water repellent cellulosic textile (Paper I) ...... 14 4.1.1 Characterization of betulin-terephthaloyl chloride (TPC) copolymer ...... 14 4.1.2 Film preparation ...... 15 4.1.3 Material preparation ...... 17

4.2 Cellulosic textiles with hydrophobic and antibacterial properties (Paper II) ...... 21 4.2.1 Oxidation and esterification ...... 21 4.2.2 Surface morphology and elemental analysis ...... 22 4.2.3 Wettability ...... 24 4.2.4 Antibacterial properties ...... 25 4.2.5 Mechanical properties ...... 28 5 Conclusions ...... 29 6 Future work ...... 30 7 Acknowledgement ...... 31 8 References ...... 32

Introduction 1

1 Introduction The use of cellulosic textiles can be traced back to 3000 B.C1, and human beings have always considered cotton, the purest cellulose source in nature, to be a main material to weave textiles, which can further be made into clothes, bags or sheets. Economic convenience, softness and sustainability have meant that cellulosic textiles are more advantageous than textiles made from synthetic fibers. However, in some cases when water repellency is needed (e.g., self-cleaning coatings, rainwear and bandages), a cellulosic textile is not a desired choice since it can easily be wetted by water 2, 3. The hydrophobization of cellulosic textile materials has therefore become a challenge as well as a topic attracting considerable attention. The commonly adopted solution to achieve hydrophobicity is to introduce wax or paraffin to lower the surface energy. This method may be easy and affordable but it introduces issues such as lack of air permeation, low comfort 4 and, most seriously, environmental pollution due to the contaminated wastewater generated during the treatment process 5. We were thus motivated to seak an environmentally friendly alternative to develop a hydrophobic cellulosic textile, where substances isolated from natural resources were considered to be a good choice.

Betulin, a natural material extractable as a white powder from the outer bark of birch 6, 7, has a structure with a hydrocarbon backbone which makes it a hydrophobe. Currently a large amount of betulin is incinerated together with birch bark to provide thermal energy, and this is ranked as the lowest application according to the biomass value pyramid 8. Using betulin to develop new materials is therefore not only environmentally friendly, but is also a good example of utilizing a side-stream product in a value-added application. In the work described in this thesis, betulin, has been used to cause changes including but not limited to the wettability of cellulosic textiles.

In Paper I, in order to explore the possibility of using betulin as a hydrophobe to improve the water repellency of textiles, the physical introduction of betulin or a betulin-based copolymer onto cellulosic textile surface by impregnation and compression molding methods was studied.

In Paper II, the possibility of covalently attaching betulin onto cellulosic textiles was explored.

2 Background

2 Background 2.1 A brief introduction to wood chemistry

Cellulose, hemicellulose, lignin and extractives are the four main components of wood cell walls 9.

2.1.1 Structure and property of cellulose As the most abundant organic polymer on earth, cellulose has been used as raw material for many applications (e.g., paper and textiles) for a long time 10. Green plants are the most common source where cellulose can be found and especially in cotton, where the cellulose content is as high as 90% 11. Cellulose consists of repeated glucose residue units. It is a linear polymer normally contains more than 10000 β-glucopyranoside residues linked via β (1-4) glycosidic bonds 10. Besides the glycosidic bonds, hydrogen bonds between adjacent glucose residues also play a role in stabilizing the structure of the chain. As can be seen in Scheme 1, hydrogen bonds “C6 oxygen - C2 hydroxyl” and “C5 oxygen – C3 hydrogen” stabilize the linear cellulose chain, while the hydrogen bonds “C6 hydrogen – C3 oxygen” connect the cellulose chains into a sheet. These sheets are then stacked via Van der Waals bonds and 휒 interactions to form cellulose fibrils, which further aggregate and are embed with hemicellulose and lignin to form woody fiber walls 12. Cellulose contains ordered and less ordered structures, and the latter allow cellulose to be degraded into crystalline cellulose with a DP of only 200 by an acid- hydrolysis process 13. The less ordered or amorphous regions also make cellulose chemically reactive, due to the free hydroxyl groups on C2, C3 and C6. Many classic cellulose-based reactions, such as TEMPO-mediated oxidation 14, are based on the reactivity of the primary hydroxyl groups on C6.

Scheme 1 Intramolecular and intermolecular hydrogen bonds in cellulose

Background 3

2.1.2 Extractives from birch bark The bark of trees consists of inner bark and outer bark, and the outer bark contains wood extractives, which are solvent extractable and suberin, which is a non-soluble natural polymer 15. The outer bark of birch contains various kinds of extractives including a high content of triterpenes 7, 16. In Betula pendula, one of the most common birch species, the extractives in the outer bark consist of 78.1% betulin, 7.9% lupeol, 4.3% betulinic acid (Scheme 2) and some other triterpenes 16.

Scheme 2 Main triterpenes in the outer bark of birch

Betulin is the major substance in the extractives. The role of betulin in the birch tree is still unclear, but the white color of birch bark is given by betulin 16. The extraction of betulin has been studied for a long time and the most common technique is solvent extraction using a Soxhlet extractor with various organic solvents (e.g., ethanol or heptane) 17. A method using supercritical carbon dioxide 18 (SC-CO2) has also been reported . Due to environmental considerations, the extraction of betulin with a green solvent and methods using pine monoterpenes and limonene have also been evaluated 19. Betulin has always been considered as a precursor for medical and pharmaceutical production since it shows positive bioactivity, such as anticancer, anti-HIV, antifungal and antibacterial properties 6, 18, 20.

2.2 Textile hydrophobization

2.2.1 A brief introduction to wetting theory On an ideal surface which is atomically flat, the wetting by a water droplet can be described by Young’s model (Scheme 3), with the equation 21: cos θY = (γSV – γSL)/ γLV [1] where θY is the contact angle of the water droplet on that surface, and γSV, γSL and γLV are the solid-vapour, solid-liquid and liquid-vapour interfacial surface tensions, respectively. 4 Background

Scheme 3Young’s model

However, an ideal smooth surface cannot be found in reality, and the surface roughness has to be considered. In 1936, Wenzel presented a model of a rough but chemically homogeneous surface that was completely wetted by a water droplet, as shown in Scheme 4. The wetting behavior of the water droplet was described by the Wenzel equation 22: cos θw = r cos θY [2] where θw is the apparent contact angle of the water droplet on that surface and r is the surface roughness, calculated as the ratio of the wetted surface area to its projected area.

Scheme 4 Wenzel model

When air is trapped under the water droplet, the Wenzel model is no longer valid and Cassie and Baxter’s model is then relevant. In 1944, Cassie and Baxter established a classic model describing the situation for a water droplet on a rough and composite surface of solid and air (Scheme 5), given by the equation 23: cos θCB = rf cos θY + f -1. [3] where θCB is the apparent contact angle on the composite surface. r is the roughness of the top of the peaks which touch the water, i.e. r is not the roughness of the whole surface 24, and f is the projected area of the rough tops of these peaks divided by the whole projected area covered by the water droplet. When f =1, this equation is the same as the Wenzel model. The precondition for this model is that the water Background 5

droplet is totally lifted by the peaks on the surface without filling the intermediate grooves. More strictly, the surface of the water in contact with air trapped in the grooves should be flat. Obviously, the greater the fraction of air trapped in the grooves the higher is the apparent contact angle since the instinctive contact angle of water with air is 180°.

Scheme 5 Cassie–Baxter model

2.2.2 Methods to obtain hydrophobic cellulose textile fibers Due to the demand for techniques such as self-cleaning 25, 26, anticorrosion 27, 28 and oil−water separation 29, 30, hydrophobic/superhydrophobic surfaces have gained great attention in recent decades. Hydrophobic/superhydrophobic surfaces need a combination of high roughness and low surface tension, and this rule also applies to textile surfaces. Research focusing on hydrophobic/superhydrophobic textiles has become a hot topic because textiles, which serve as a substrate, possess many advantages such as low density, inexpensiveness and breathability 31. Besides the simple introduction of paraffin waxes onto textiles, various other attempts to prepare hydrophobic textiles have been reported. Electrospinning was used to generate textile fibers based on poly(caprolactone) and poly(styrene-block- dimethylsiloxane) block copolymer with low surface energy 32. The sol-gel method was also widely used to form a low-surface-energy fluorinated coating on textile to achieve hydrophobization 33, and fluoroamine or alkylamine can be grafted onto a nylon polymer surface to give it hydrophobic properties 24. Dankovich et al reported the application of a transesterification reaction to functionalize cotton textile with fatty acid groups from triglycerides, to generate a hydrophobic textile 34. In order to use more biobased hydrophobization agents, natural extracts from outer bark of birch was used to obtain hydrophobic fiber surfaces and the methods of dip- coating, film coating and grafting were performed and evaluated in this thesis.

2.3 Nucleophilic acyl substitution

The reaction between an acyl halide and an alcohol is usually catalyzed by a nucleophile such as pyridine or trimethylamine 35 with a higher basicity than the 6 Background

alcohol itself. In the present work, pyridine is selected as the catalyst and the mechanism is given in Scheme 6. Due to its higher basicity, pyridine (1) will make a nucleophilic attack on the acyl halide before alcohol to form a positively charged intermediate III by releasing the halide ion group. This intermediate is highly electrophilic and rapidly undergoes attack from the alcohol to form V. V is soon deprotonated and another pyridine molecule “pyridine (2)” then binds to the liberated proton to form a complex cation, moving the reaction equilibrium to the right. Pyridine in this reaction thus also plays a role as an acid-binding reagent. After deprotonation releasing the ester and pyridine 36, the catalytic cycle is completed.

Scheme 6 Mechanism of pyridine-catalyzed nucleophilic acyl substitution

2.4 TEMPO-mediated oxidation

(2,2,6,6-Tetramethylpiperidin-1-yl)oxyl, known as TEMPO, is widely used as mediator in oxidation reactions known as TEMPO-mediated oxidation. These reactions are usually applied to oxidize the primary hydroxyl groups in cellulose to carboxyl groups 37. Scheme 7 illustrates the reaction route employing the TEMPO/ NaClO/NaClO2 system under weak acidic conditions (pH 4.8). Briefly, TEMPO is first oxidized by NaClO to N-oxoammonium ion, which then oxidizes the primary hydroxyl group to an aldehyde group, forming hydroxylamine. The aldehyde group is then oxidized to a carboxyl group by NaClO2, which is in turn reduced to NaClO. This NaClO reoxidizes the hydroxylamine to N- oxoammonium ion, forming NaCl, to complete the reaction cycle 37.

Background 7

Scheme 7 The TEMPO/ NaClO/NaClO2 system to oxidize primary hydroxyl groups to carboxyl groups

In Paper II, carboxyl functionalized cellulosic textile is created according to the above reaction route.

2.5 Aim and objectives

The aim of the present work is to prepare functional cellulosic textile fibers using a natural substance such as betulin, and to propose that betulin can be used in a value- added application instead of being simply incinerated with bark as solid fuel. 1) To develop a cellulosic textile using betulin to achieve hydrophobic and water- repellent properties. The static water contact angle of the prepared textile shall be as high as possible (at least 90°) and a water repellency score according to AATCC standard higher than 70. 2) To explore the possibility of covalently attaching betulin onto a cellulosic textile surface to improve in some properties, such as hydrophobicity. 8 Experimental

3. Experimental 3.1 Materials

Cellulosic textile: commercial cotton textile (Hemtex, Stockholm, Sweden) made of 100% cotton was used as a cellulose source. Reagents (used as received without further purification): chloroform (99.9%), terephthaloyl chloride (TPC, ≥99%), tetrahydrofuran (THF, 99.99 %), sodium hydroxide (NaOH), toluene (≥99.5%), phenolphthalein, sodium hypochlorite solution (NaClO, 10%), 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO, 98%), sodium chlorite (NaClO2, 80%), acetic acid (CH3COOH, ≥99.7%), sodium acetate anhydrous (CH3COONa, ≥99%), sodium borohydride (NaBH4, ≥98%), glutaraldehyde (50 wt%) and 1 M hydrochloric acid (HCl) (Sigma-Aldrich); ethyl acetate (99.98%) and pyridine (>99%) (Fisher Scientific); sodium hydroxide (NaOH), ethanol (96%) (VWR International); L (+)-tartaric acid (Scharlau Chemicals). Dye: Green food-coloring dye (Dr. Oetker Sverige AB, Stockholm, Sweden). Betulin: Betulin was extracted from ground birch (Betula verrucosa) outer bark by Soxhlet extraction for 48 h. The bark was collected in a park near KTH Royal Institute of Technology (Stockholm, Sweden). After solvent evaporation and drying, betulin was obtained as a white powder with a purity of ca. 80%, as 1 1 determined by H NMR. H NMR (CDCl3) δ (ppm): 4.70 (s, 1H, 29a‐H), 4.60 (s, 1H, 29b‐H), 3.83 (d, 1H, 28b‐H), 3.36 (d, 1H, 28a‐H), 3.21 (dd, 1H, 3‐H), 2.41 (m, 1H, 19‐H), 1.70 (m, 3H, 30‐H), 1.67‐0.70 (m, 40-H).

3.2 Pretreatment of cellulosic textile

An Electrolux EWC1350 laundering machine was used to wash the textile, which was cut into 18 cm×18 cm pieces, at 40°C for 2h to remove impurities. A non- bleaching washing powder was added. The textiles were then washed with 2 g L-1 NaOH solution for 90 min at 98°C before being thoroughly washed in distilled water. Finally, the textiles were dried at 105°C overnight 38.

3.3 Preparation of a water-repellent cellulosic textile by impregnation and compression molding (Paper I)

The betulin-TPC copolymer was bulk synthesized by mixing betulin and TPC in a molar ratio of 1:3 with excess pyridine in toluene (concentration of betulin in toluene is 0.15 mol L-1). The mixture was heated to 105°C and maintained anhydrously for 24 h, followed by cooling to ambient temperature. After solvent evaporation, washing with 1 M HCl, recrystallization in ethanol and drying at 60°C overnight, a yellowish betulin-TPC copolymer was obtained and analyzed by 1H 1 NMR. H NMR (CDCl3) δ (ppm): 8.13 (s, 4H, Car‐H), 4.75 (s, 1H, 29a‐H), 4.65 (s, Experimental 9

1H, 29b‐H), 4.58 (d, 1H, 28b‐H), 4.15 (d, 1H, 28a‐H), 3.76 (dd, 1H, 3-H), 2.55 (s, 1H, 19‐H), 1.88‐0.94 (m, 42H, Csp3‐H). Three types of betulin solutions and a betulin-TPC copolymer solution were prepared, as shown in Table 1.

Table 1 Preparation of betulin and betulin-TPC copolymer solutions Solute Solvent Concentration (g L-1) 3.75 Ethanol Betulin 7.5 Ethyl acetate 7.5 Betulin-TPC THF 3.75 copolymer

The textile samples were impregnated in solutions, as shown in Table 1; in ethanol solutions of betulin at concentration of 3.75 g L-1 and 7.5 g L-1, to prepare materials A1 and A2; in an ethyl acetate solution of betulin at a concentration of 7.5 g L-1, to prepare material A3; in a THF solution of betulin-TPC copolymer, to prepare material B. Betulin-based films (films 1-6) and Betulin-TPC-copolymer-based films (films 7- 11) were prepared by solvent casting from mixtures with compositions shown in Table 2. A pure CTA film was prepared as a reference sample.

Table 2 Mixtures of cellulose triacetate (CTA) with betulin or betulin-TPC copolymera CTA Betulin Mixture/film Copolymer (mg) Ratiob (mg) (mg) 1 290 116 - 1:0.4 2 290 203 - 1:0.7 3 290 290 - 1:1 4 290 377 - 1:1.3 5 290 461 - 1:1.6 6 290 551 - 1:1.9 7 290 - 116 1:0.4 8 290 - 203 1:0.7 9 290 - 290 1:1 10 290 - 377 1:1.3 11 290 - 461 1:1.6 a6 ml of chloroform was used to dissolve each mixture; bMass ratio of CTA to betulin or betulin-TPC copolymer. The film with the greatest hydrophobicity of films 1-6 (Table 2) and the film with the greatest hydrophobicity of films 7-11 (Table 2) were coated on textile samples separately under a pressure of 80 kN at 10 Experimental

210°C for 4 min by a Fontijne TPB compression molding instrument, to form materials C and D, respectively.

3.4 Preparation of hydrophobic and antibacterial cellulosic textile by surface chemical modification (Paper II)

Cellulosic textile (3 g) was impregnated with an acetate buffer at pH 4.5 (150 ml, 0.05M) together with TEMPO (60 mg), NaClO2 (150 mg) and NaClO solution (9.3 ml, 10%). The mixture was maintained at 60°C for 15, 45 or 180 min to obtain oxidized textiles, which were then acid-washed with HCl solution (pH2) followed by a thorough washing with deionized water to obtain carboxyl-functionalized textiles denoted 15COOH, 45COOH and 180COOH, respectively, as shown in Table 3. Oxidized textile (100 mg) of each type was impregnated with solution of betulin in toluene (10 ml, 0.16 mol L-1) together with tartaric acid (25 mg). The mixture was maintained at 110°C for 6, 24 or 72 h to obtain betulin-grafted textiles, as shown in Table 3. The samples obtained were Soxhlet extracted first with THF and then water to remove non-attached chemicals, followed by air drying overnight.

Table 3 Preparation of oxidized textiles and betulin-grafted textiles by oxidation and esterification Duration of Duration of Denotation oxidation (min) esterification (h) Reference textile Ref. cotton - - 15COOH 15 - Oxidized textile 45COOH 45 - 180COOH 180 - 15COOB6 15 6 15COOB24 15 24 15COOB72 15 72 45COOB6 45 6 Betulin-grafted 45COOB24 45 24 textile 45COOB72 45 72 180COOB6 180 6 180COOB24 180 24 180COOB72 180 72

3.5 Characterization

3.5.1 Nuclear magnetic resonance (NMR) 1H NMR spectra were obtained using a Bruker UltraShield 400 MHz spectrometer (Germany), using deuterated chloroform (CDCl3) as solvent.

Experimental 11

3.5.2 Fourier transform infrared spectroscopy (FTIR) FTIR spectra were obtained using a Perkin-Elmer Spectrum 2000FT-IR. 32 scans between 4000 and 600 cm-1 were obtained for each spectrum.

3.5.3 Size exclusion chromatography (SEC) A Viscotek TDA model 301 system attached with two LT4000L columns (7.8×300 mm) was used to perform SEC. THF was used as solvent and the sample concentration was 2 g L-1.

3.5.4 Differential scanning calorimetry (DSC) A Mettler Toledo DSC 820 calorimeter connected with a cryocooler and a sample robot was used to perform DSC. The samples were heated from 25°C to 300°C, followed by cooling to -30°C before being reheated to 300°C in a N2 atmosphere with a flow rate of 50 ml min-1.

3.5.5 Scanning electron microscopy (SEM) SEM images were obtained by a Hitachi S-4800 Field-emission scanning electron microscopy. The samples were sputtering coated with a 4 nm gold layer before imaging.

3.5.6 Energy dispersive X-ray analysis (EDS) An Inca Oxford EDS instrument (X-MAX, N80) attached to the SEM instrument was used to obtain EDS spectra with an accelerating voltage of 15 kV. All the data were analyzed by AZtec INCA software.

3.5.7 Static water contact angle (SWCA) measurement A KSV Instruments CAM 200-meter equipped with a Basler A602f camera was applied to measure the contact angles. The contact angle of water on each sample was calculated as the average of the values of 5 different positions.

3.5.8 AATCC water repellency test The extent of water repellency was determined by a standard spray test: AATCC- TM22 39. In general, 250 mL of water was sprayed onto the surface of a textile sample tautened by a specific tester under controlled conditions. After spraying, the wetting pattern on the textile sample was compared to pictures in a standard rating chart, as listed in Table 4, to obtain a score which indicated the extent of water repellency.

12 Experimental

Table 4 AATCC spray test rating standards Rating Evaluation 100 No sticking or wetting of the specimen face 90 Slight random sticking or wetting of the specimen face 80 Wetting of specimen face at spray points 70 Partial wetting of the specimen face beyond the spray points Complete wetting of the entire specimen face beyond the spray 50 points 0 Complete wetting of the entire face of the specimen

3.5.9 Carboxyl content (CC) determination Oxidized textile (500 mg) was impregnated in a solution of CH3COONa (30 ml, 0.5M) in deionized water (50 ml) and the mixture was constantly shaken for 2 h, after which up to 30 ml of the liquid was titrated with 0.01M NaOH. The measurement was triplicated and the CC was calculated according to the equation: 80 ×0.01 푀×V(NaOH) 30 COOH = w [4] m×(1− ) 100 where V(NaOH) stands for the volume (mL) of NaOH solution used and m and w are the mass (g) and moisture content (%) respectively of the impregnated sample.

3.5.10 Degree of oxidation (DO) DO was calculated according to the equation: 162×CC DO = [5] 1−14×CC where CC is the carboxyl content as described above, 162 is the molecular weight (Da) of a glucopyranose unit in cellulose, 14 is the difference in molecular weight (Da) between a carboxyl group and a CH2OH group.

3.5.11 Tensile test An Instron universal testing machine 5944 (UK) with a 500 N load cell was used to perform the tensile tests. The initial gap between two grips was 10 mm and the grips were separated at a rate of 1% s-1 at a temperature of 23°C and humidity of 50% to determine the breaking tensile strength and elongation at break of each sample. At least three specimens from each sample were tested and the mean value and standard deviation were calculated.

3.5.12 Antibacterial assays A nutrient broth (Difco, Stockholm, Sweden) was used to incubate Gram-negative bacteria Escherichia coli (E. coli) K-12 and Gram-positive Staphylococcus aureus (S. aureus) (BioRad, Solna, Sweden) at 37°C for 18 h with constant shaking. A bench-top centrifuge (VWR, Stockholm, Sweden) (5000 rpm, 5 min) was used to harvest the cells. Bacterial removal and bacterial growth inhibition were used to examine the antibacterial effect: Experimental 13

Bacterial removal test: original textile (8 mg) or betulin-grafted textile (15COOB24, 45COOB72 or 180COOB24, 5 mm  15 mm), and “+” control were place in Eppendorf mini tubes with a bacterial concentration of 2  106 CFU ml-1, followed by constant shaking at 37°C for 18 h in an incubator. Thereafter, 0.2 ml of solution was withdrawn and diluted in 1.8 ml, 5.4 ml and 7.2 ml of ¼ Ringer’s solution separately, before 1 ml of each diluted solution was dropped onto a piece of PetrifilmTM (3M, Sollentuna, Sweden). ImageJ was then used to count the bacterial colony.

Bacterial growth inhibition: After the bacterial removal test, each textile-bacteria suspension was mixed with nutrient broth (0.2 ml), followed by constant shaking for 18 h at 37°C in an incubator. A MultiSkan FC microplate spectrophotometer (Thermo Scientific, Stockholm, Sweden) was used to estimate the amount of bacteria by determining the optical density (OD) of each suspension at a wavelength of 620 nm. The true viability was also determined by counting the amount of bacterial colonies on the Petrifilm.

14 Results and Discussion

4 Results and Discussion 4.1 Impregnation and compression molding to prepare water repellent cellulosic textile (Paper I)

Betulin or a betulin-based copolymer synthesized from betulin and TPC was introduced onto the textile surface by solution impregnation or film compression molding. The copolymer was characterized to verify the success of the reaction, and the surface morphology, wettability and water repellency of the prepared materials were evaluated.

4.1.1 Characterization of betulin-terephthaloyl chloride (TPC) copolymer Betulin-TPC copolymer was synthesized according to the method described in 3.3. Figure 1 shows the appearance of both the betulin and the betulin-TPC copolymer.

Fig 1 Powder of betulin (left) and betulin-TPC copolymer (right)

The synthesized copolymer had a glass transition temperature (Tg) of 253°C, according to DSC curve (Figure 2); a Mw of 9000 Da and PDI of 3.1, according to SEC.

Fig 2 DSC curve from -30°C to 300°C for betulin-TPC copolymer Results and Discussion 15

Figure 3 shows the FTIR spectra of betulin and betulin-TPC copolymer, normalized -1 with respect to the peak at 1645 cm corresponding to C=CH2 vibration. A broad band corresponding to hydroxyls was evident from 3650 cm-1 to 3200 cm-1 in the spectrum of betulin but not in that of the betulin-TPC copolymer, which indicates that betulin was largely consumed by the reaction. A strong band at 1716 cm-1 from the copolymer was due to the stretching vibration of the C=O group and, together with the bands at 1264 cm-1 and 1100 cm-1, this indicate that the creation of ester bonds was successful.

Fig 3 FTIR spectra of betulin and betulin-TPC copolymer

4.1.2 Film preparation Figure 4 shows the betulin-based films and betulin-TPC-copolymer-based films as well as the reference film of pure CTA. In the betulin and betulin-TPC copolymer based films, the film surface became increasingly less homogeneous with an increasing proportion of betulin or betulin-TPC copolymer. This may be because the poor solubility of betulin or of the betulin-TPC copolymer allows the undissolved solute to aggregate to particles which are non-uniformly distributed on the film surface, or because the viscosity of the mixture increases with increasing concentration of solute and allowing air to be trapped inside and form dots on the film surface. 16 Results and Discussion

Fig 4 1-6) Betulin-based film 1-6; 7-11) Betulin-TPC-copolymer-films 7-11; R) Reference film

Figure 5 shows the water contact angle of each film. The different concentrations of solute lead to films with different wettability values, but for a given concentration of solute, the betulin-based film exhibit in a higher contact angle than the betulin- TPC-copolymer-based film. Film 4 showed the greatest hydrophobicity of the betulin-based films with an average contact angle of ca. 125°, and film 9 showed the greatest hydrophobicity of the copolymer-based films with an average contact angle of ca. 100°. Film 4 and 9 were therefore used for coating onto textiles to prepare water repellent materials.

Results and Discussion 17

Fig 5 Water contact angle of a) films 1-6, b) films 7-11 and reference film as a function of time

4.1.3 Material preparation Six different materials were prepared according to the methods listed in Table 5.

Table 5 Material preparation method Material Preparation method A1 textile impregnated with 3.75 g L-1 ethanol solution of betulin A2 textile impregnated with 7.5 g L-1 ethanol solution of betulin A3 textile impregnated with 7.5 g L-1 ethyl acetate solution of betulin B textile impregnated with 3.75 g L-1 THF solution of betulin-TPC copolymer C textile coated with betulin-based film (film 4, CTA: betulin=1:1.3) D fabric coated with betulin-TPC-copolymer-based film (film 9, CTA: betulin-TPC copolymer=1:1)

Figure 6 shows the surface morphologies of materials A1, A2, A3, B and a reference textile. Betulin particles, which are thin cylinders in shape, can be observed but not uniformly distributed on A1, A2 and A3. In b) material B, pieces of betulin-TPC-copolymer wrapping the textile fiber can be seen. The d) reference textile fiber shows only folds but no adsorbed particles. 18 Results and Discussion

Fig 6 SEM images of the surfaces of a) A1, c) A2, e) A3, b) B and d) reference textile.

Figure 7 shows a-d) the coating surface and e, f) the cross section surface of a, c, e) material C and b, d, f) material D. The textured surface of a) material C may due to the exposure of textile fibers underneath caused by film cracking during the compression molding process. However, b, d) material D presents a smooth surface. The materials were fractured in liquid nitrogen and the cross sections of c) C and f) D were also observed. The boundary dividing film and textile fibers can easily be identified, indicating that there is no interpenetration between film and textile substrate. Results and Discussion 19

Fig 7 SEM images of the surfaces of material C (a, c, e) and material D (b, d, f); (a, b) 30x top, (c, d) 400x top, (e, f) 700x cross section

Figure 8 shows water contact angle of each material as a function of time. The mean contact angle for each material averaged over 3 min is 153° (A2), 151° (B), 149° (A3), 147° (A1), 123° (C) and 104° (D). Of these materials, material A2 exhibited the greatest hydrophobicity while material B and A3 exhibited slightly lower contact angles. Materials A2 and B reached the definition of superhydrophobicity, which requires a static water contact angle greater than 150°. The low surface energy of betulin and the roughness provided by the hairy textile fibers generally result in a hydrophobic surface. On the other hand, although the contact angle of C and D are not close to 150°, the film coating method changed the textiles from hydrophilic to hydrophobic, as the contact angles on both C and D were greater than 90°. 20 Results and Discussion

Fig 8 Water contact angle of materials A1, A2, A3, B, C and D as a function of time

A widely used water repellency method “AATCC TM-22” was also applied 40. The result is shown in Figure 9. Materials A2 and B presented a pattern of annular water droplets surrounding the central area, which shows little water repellency like that of the reference sample. However, the surrounding parts can be scored as 70, according to the rating chart in 3.5.8. On the other hand, Materials C and D showed uniformly distributed water droplets and a score of 80 can be given to them. No low score regions were seen in the test areas of materials C and D.

Fig 9 Wettability and water repellency; a-e) profile of water droplet in contact angle measurements, f-j) materials and the reference textile, k-o) materials and the reference textile after spray test Results and Discussion 21

4.2 Cellulosic textiles with hydrophobic and antibacterial properties

(Paper II)

Betulin was covalently attached to cellulosic textile, and TEMPO-mediated oxidation and tartaric acid-catalyzed esterification were performed to optimize the preparation.

4.2.1 Oxidation and esterification The strong band at 1727 cm-1 of 15, 45 and 180COOH shown in Figure 10a) is associated with C=O stretching 41 and this confirms the success of the oxidation. Figure 10d) shows the CC and DO of corresponding samples. Both the FTIR results and CC determinations indicate that the oxidation was completed. 180COONa, which is the oxidized product of Ref. cotton prior to the acid wash, together with 180COOH and Ref. cotton were analyzed by FTIR, as shown in Figure 10b). The shift of the C=O band from 1604 cm-1 to 1727 cm-1 after acid wash indicates that the conjugation effect of the –COO- group no longer existed. This phenomenon could be used to identify the ester bonds created since the C=O band of the ester bond overlapped with the C=O band from the unconsumed carboxyl groups at ca. 1730 cm-1 42. These betulin-grafted textiles had been alkali treated to isolated the created ester bonds, which should be preserved from hydrolysis. Weak alkali conditions were therefore applied for a short time period: NaOH solution at pH10 was used to treat those betulin-grafted samples at room temperature for 1 h. 180COONa and the betulin-grafted samples were then subject to FTIR analysis, as shown in Figure 10c). The bands assigned to ester bonds were clearly evident at 1730 cm-1 after alkali treatment, suggesting that the esterification was successful. In addition, the higher infrared peak indicates a larger amount of ester bonds within the sample.

22 Results and Discussion

Fig 10 a) FTIR spectra of Ref. cotton and its 15-min, 45-min, and 180-min oxidized products; b) FTIR spectra of Ref. cotton and its 180-min oxidized product before (-ONa) and after (-OH) acid wash; c) FTIR spectra of all 9 betulin-grafted textiles after alkali wash together with 180COONa; d) Carboxyl content (CC) and degree of oxidation (DO) of samples in (a)

4.2.2 Surface morphology and elemental analysis Figure 11 shows the surface morphology after each step. Ref. cotton, the original textile, shows only folds on the fibers (Figure 11a) and this corresponds with the characteristics of pristine cellulosic textile fibers 43, 44. After oxidation, the surface of the textile was the same as before and no obvious damage was found, suggesting that the oxidation reaction was mild. The esterification was however relatively harsh since burls on a nanoscale can be found in the zoom-in area of Figure 11c, which shows the sample after a long esterification process. In addition, a sample was prepared by impregnating Ref. cotton in a 0.16 mol L-1 toluene solution of betulin to physically adhere betulin particles onto the fiber surfaces, as shown in Figure 11d, where free betulin particles have a thin cylinder shape, as reported in paper I 45. Since no such particles can be seen in Figure 11c, it can be concluded that the non-attached betulin was to a large extent removed by Soxhlet extraction. The complete removal of non-attached betulin is important in EDS analysis since it can indicate that the detected betulin comes only from covalently attached betulin and therefore further confirms the success of the esterification. Covalently attached Results and Discussion 23

betulin can be detected by comparing Figure 12c with Figure 12b. The C/O ratio increased to a great extent after the esterification, indicating that betulin, which possesses an abundance of C elements but few O elements, was covalently attached. The combination of SEM images and EDS spectra therefore also confirms the esterification.

Fig 11 SEM images of a) Ref. cotton; b) 180COOH; c)180COOB72; d) Ref. cotton impregnated in 0.16 mol L-1 toluene solution of betulin 24 Results and Discussion

Fig 12 EDS spectra of a) Ref. cotton; b) 180COOH; c)180COOB72

4.2.3 Wettability Of the nine samples, only sample 45COOB72, 180COOB24, 180COOB72 and 15COOB72 presented measurable contact angles, and the wettability of these four samples is shown in Figure 13. In Figure 13a), the contact angle of water on 15COOB72 gradually drops from 128° to 77°, indicating that the sample is hydrophobic for only a few seconds and thus cannot be classified as a hydrophobic material. The other three samples (45COOB72, 180COOB24 and 180COOB72) exhibited a stable contact angle over time (Figure 13a) with a mean value of ca. Results and Discussion 25

130° (Figure 13b), suggesting that stable hydrophobicity had been achieved. It was concluded from the SEM images that the non-attached betulin had been mostly removed. The stable hydrophobicity can therefore be attributed to the covalently attached betulin moieties and this deduction is also in agreement with the EDS result.

Fig 13 a) static contact angle of 45COOB72, 180COOB24, 180COOB72 and 15COOB72 as a function of time; b) mean contact angle of 45COOB72, 180COOB24, 180COOB72 and 15COOB72 averaged over 2 min

4.2.4 Antibacterial properties The amount of viable bacteria after 18h-incubation with different samples (Ref. cotton, 15COOB24, 45COOB72 and 180COOB24) with (Figure 14b) and without (Figure 14a) nutrients is given in Figure 14. The pure cellulosic textile sample did not show any bacterial reduction but even enhanced the proliferation of both kinds of bacteria, indicating that the polysaccharide-based textile act as a nutrient, as in similar cases where cellulosic pulp fibres served as nutrients 46. The betulin-grafted samples, however, showed a bacterial reduction of more than 90%. 180COOB24, in particular, exceeded a bacterial reduction of 99%. In Figure 14b where nutrients are accessible, the reference textile showed a greater bacterial growth enhancement than in Figure 14a.

26 Results and Discussion

Fig 14 Concentration (CFU/mL) of E. colo and S. aureus after 18 h incubation with different textile samples a) with or b) without added nutrients; + stands for “plus control” which contained no textile sample; The table below shows the bacterial reduction percentage

Figure 15 shows the general density and distribution of bacteria as red dots on Petrifilms on which small piece of textile were placed. Compared with the reference textile, sample 180COOB24 resulted in a reduction for both kinds of bacteria as the white colour of the Petrifilm is largely exposed from the red color which indicates the presence of bacteria.

Fig 15 Petrifilms contain E. coli or S. aureus solution incubated for 18 h with reference textile or 180COOB24

The textile samples were also observed by SEM, as shown in Figure 16. More bacteria can be seen on the reference textile than on the 180COOB24 sample. Results and Discussion 27

Fig 16 SEM images of a) E. coli growth on reference textile, b) E. coli growth on 180COOB24, c) S. aureus growth on reference textile and d) S. aureus growth on 180COOB24

It has been reported that a low pH leads to low bacterial viability 47, 48. To exclude the influence of pH, samples 15COOB24, 45COOB72 and 180COOB24 were incubated at 37°C in Ringer’s solution. After 18 h, no pH change was observed and the pH was ca. 7 for all samples (Figure 17). The influence of pH can therefore be excluded from this study.

Fig 17 The pH of Ringer’s solution contains samples 15COOB24, 45COOB72, 180COOB24 or reference textile after 18h incubation at 37°C. 28 Results and Discussion

4.2.5 Mechanical properties Table 6 presents the tensile properties of the materials. The tensile strength showed a rapid reduction due to the oxidation process, in agreement with previous studies 49. The mechanical properties also decreased slightly with increasing esterification time, probably due to the depolymerization of cellulose caused by the hydrolysis catalyzed by tartaric acid at elevated temperature.

Table 6 Tensile strength and elongation at break of Ref. cotton, oxidized textiles and betulin-grafted textiles Elongation at break Sample Tensile strength (MPa) (%) Ref. cotton 30.5±2.0 21.6±8.0 15COOH 19.9±1.8 14.0±0.5 45COOH 7.8±0.6 24.8±0.6 180COOH 6.6±0.3 22.2±0.5 15COOB24 5.7±0.5 22.6±1.3 15COOB72 4.5±0.7 21.6±0.7 45COOB24 5.7±0.7 10.1±0.4 45COOB72 2.9±0.5 8.3±0.6 180COOB24 4.2±0.8 19.3±0.9 180COOB72 2.2±0.2 17.9±0.6

Conclusions 29

5 Conclusions Betulin isolated from birch bark can be used to enhance the water repellency and antibacterial properties of cellulosic textile. Different methods of introducing betulin onto textile were explored. Cellulosic textiles were modified by being coated with betulin monomer particles, with a betulin-based copolymer, or films prepared from betulin or the betulin-based copolymer. With this modified textile, a static water contact angle of 153° and a water repellency score of 80 were obtained. A betulin-grafted textile was prepared by esterifying betulin with a carboxyl-functionalized textile, and with this modified textile a contact angle of 136°, and a bacterial removal of more than 99% were reached. In summary, this thesis proposes a concept that betulin can be used in a value-added application (e.g., functional materials), rather than being simply incinerated with bark as solid fuel to provide thermal energy. This concept is also in line with the concept of biorefinery.

30 Future work

6 Future work The material preparation used chemicals like TEMPO, terephthaloyl chloride, THF and pyridine that are not environmentally friendly. Less toxic chemicals can hopefully be found to replace them. The flexibility and air permeability of the films used as coating in Paper I need to be tested. The mechanism of bacterial removal by betulin needs to be investigated. The durability against home-washing of prepared functional textile needs to be tested. Acknowledgement 31

7 Acknowledgement I would first like to thank my main supervisor Prof. Monica Ek, for accepting me as a PhD student, training me to become an independent researcher and giving me opportunities to integrate into the Swedish and Chinese forest industries. I believe without doubt that I shall benefit from the experience of being a member of the division of wood chemistry and pulp technology in the future. I would also like to express my great appreciation to my co-supervisor Dr. Dongfang Li for his particular guidance in my experimental work, detailed teaching of theories that I was not familiar with and adequate research planning that suits me well. Chao Chen as co-author in the second paper is deeply thanked for the contribution to the antibacterial test, which opens a new door in my research area. Apart from research, I also appreciate that he helps me in many aspects as a friend in my daily life. China Scholarship Council (CSC) is thanked for financial support for my doctoral study. “Knut och Alice Wallenbergs stiftelse” is thanked for providing scholarship to support me to attend international conference. I am also grateful to my colleagues in the division, department and school for giving me precious advice and help. I thank my colleagues for turning me into a skilled user of different analytical instruments: Joakim Engström, Samer Nameer, Anastasia Riazanova, Malin Nordenström, Yunus Görür and Erik Jungstedt. Last but not least, I would like to thank my family for their constant concern. 32 References

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