CONTROL OF THE MECHANICAL BEHAVIOR OF BACTERIAL CELLULOSE BY MERCERIZATION

by XINYU WU

Submitted in partial fulfillment of the requirements

for the degree of Master of Science

Thesis Advisor: Dr. Ozan Akkus

Mechanical and Aerospace Engineering

CASE WESTERN RESERVE UNIVERSITY

January 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Xinyu Wu

candidate for the Master of Science degree*.

Dr. Ozan Akkus

Committee chair，Advisor

Department of Mechanical and Aerospace Engineering

Dr. Clare M. Rimnac

Committee member

Department of Mechanical and Aerospace Engineering

Dr. Joseph M. Mansour

Committee member

Department of Mechanical and Aerospace Engineering

Defense date: 10/27/2017

*We also certify that written approval has been obtained for any proprietary material contained therein. Table of contents

List of tables ...... V

List of Figures ...... VI

Abstract ...... VIII

Acknowledgement ...... X

Chapter 1 Background ...... 11 1.1 Bacterial cellulose ...... 11 1.1.1 History ...... 11 1.1.2 Synthesis and Chemistry of Bacterial cellulose ...... 13 1.1.3 Structures of Bacterial cellulose ...... 15 1.2 Culture conditions ...... 17 1.3 Applications of BC in regenerative medicine ...... 18 1.3.1 Wound dressing ...... 19 1.3.2 Artificial blood vessel ...... 21 1.3.3 Drug delivery ...... 23 1.3.4 Bone tissue engineering ...... 25 1.3.5 Cartilage ...... 27 1.3.6 Dentistry ...... 28 1.3.7 Neurosurgical Applications ...... 29 1.4 Mercerization ...... 30 1.5 Conclusion and perspective ...... 31 1.6 Motivation of this thesis ...... 33

Chapter 2 Experimental ...... 35 2. 1 Materials ...... 35 2.1.1 Raw materials and processing ...... 35 2.1.2 Removal of bacteria ...... 35 2. 2 Sample preparation ...... 36 2.2.1 Mercerization ...... 36

III 2.2.2 Preparation of patterned BC samples ...... 36 2.3 Shrinkage test ...... 39 2.4 Mechanical testing ...... 40 2.5 Digital Image Correlation (DIC) set up and analysis ...... 40 2.7 Raman spectroscopy ...... 43 2.8 X-ray Diffraction ...... 43 2.9 Statistical analysis ...... 44

Chapter 3 Results ...... 45 3.1 Effect of NaOH concentration on BC shrinkage ...... 45 3.2 2D strain distribution of mercerized BC ...... 48 3.3 Effect of NaOH concentration on mechanical properties ...... 49 3.4 Raman Spectra and XRD ...... 52 3.5 Effect of mercerized patterns on the mechanical properties of BC ...... 54 3.6 Effect of mercerized patterns on the Poisson’s ratio ...... 56

Chapter 4 Discussion and conclusions ...... 59

Appendices ...... 74 A. The fabrication of BC sheet with porosity...... 74 B. The effect of temperature of mercerization on the optical properties of BC ...... 75 C. The effect of medium culture on the yield of BC ...... 77 D. Tables of data shown in figures of this work ...... 78

References ...... 80

IV

List of Tables

Table 1.1 Mechanical properties of BC based composites…………………………………....32

Table 3.1. Mechanical properties of Mercerized BC……………………………………….....51

V List of Figures

Figure 1.1 Bacterial cellulose sample collected from our lab ...... 11 Figure 1.2 Symbiotic culture of bacteria and yeast ...... 13 Figure 1.3 The synthesis process of BC ...... 14 Figure 1.4 Cellulose types ...... 16 Figure 1.5 Different types of bioreactors designed to produce BC ...... 18 Figure 1.6 BC applied on a wound located on the hand as a dressing material ...... 20 Figure 1.7 BC tubes with different inner diameters ...... 22 Figure 1.8 Small caliber BC graft in vivo ...... 23 Figure 1.9 In vivo operation of BC membrane as as a barrier membrane on guided bone regeneration...... 25 Figure 1.10 The morphology of porous bacterial cellulose produced with paraffin wax particles of size 300–500 µm ...... 27 Figure 1.11 Dura defects were patched by BC on the left side...... 30 Figure 2.1 The patterning process ...... 36 Figure 2.2 Geometric parameters of striped patterns...... 37 Figure 2.3 Geometric parameters of the re-entrant hexagonal honeycombs structure...... 38 Figure 2.4 Re-entrant honeycomb structure expends under stretching...... 38 Figure 2.5 Unmercerized and mercerized samples...... 39 Figure 2.6 The sample prepared for the DIC analysis ...... 41 Figure 2.7 Strain measurement ...... 42 Figure 3.1 Shrinkage of BC after reacting with different concentrations of NaOH solution at room temperature ...... 46 Figure 3.2 Shrinkage of BC when treated with different NaOH concentrations ...... 46 Figure 3.3 The shrinkage of bacterial cellulose treated with 3 M KOH and 3 M NaOH ...... 47 Figure 3.4 Strain map of Eyy along the tensile loading direction ...... 48 Figure 3.5 Typical stress-strain curves of native BC and BC treated with different concentration of NaOH...... 50

VI Figure 3.6 Effect of NaOH concentration on mechanical properties of BC ...... 50 Figure 3.7 Mechanical behavior of BC samples mercerized under different temperatures ...... 51 Figure 3.8 Typical XRD spectra for untreated BC and mercerized BC films ...... 53 Figure 3.9. Typical Raman spectra for untreated BC and mercerized BC films...... 53 Figure 3.10 The effect of striped patterns on mechanical properties of BC samples...... 55 Figure 3.11 Exx strain map of a BC sample with re-entrant hexagonal honeycombs patterns ...... 57 Figure 3.12 Eyy strain map of a BC sample with re-entrant hexagonal honeycombs patterns ...... 57 Figure 3.13 Comparison of Poisson’s ratio values...... 58 Figure 4.1 The mechanism of mercerization on BC ...... 60 Figure 4.2 Scanning electron microscope figures of native BC and mercerized BC ...... 60 Figure 4.3 Comparison of FEM model and DIC result...... 67 Figure 4.4 The rupture of the BC patterned with re-entrant honeycomb structure...... 69 Figure 4.5 Auxetic structures achieved from different re-entrant geometries...... 70 Figure 4.6 FEM results of square patterned models………………………………...... 72

VII Control of the Mechanical Behavior of Bacterial Cellulose by Mercerization

By XINYU WU

Abstract

The process of mercerization of bacterial cellulose (BC) is elaborated in this work.

During the mercerization of BC films, the structural conversion and crystallinity changes from native cellulose I to cellulose II was characterized using X-ray diffraction (XRD) and Raman spectroscopy. The shrinkage of BC samples exhibited a significant increase with increasing NaOH solution concentration. Significant changes in mechanical properties of BC after mercerization were observed using Digital image correlation (DIC). The mechanical behavior of mercerized BC was tuned finely by mercerization of BC samples in NaOH solution with different concentrations.

Young’s modulus of the BC samples demonstrated a significant reduction of 30 folds by treating the BC samples with 7 M NaOH. The elongation at break increased significantly with increase in

NaOH solution concentration to 7 M. Patterns were designed to protect certain parts of the material from mercerization while the rest of material was subject to the alkaline treatment. As a result, a custom designed combination of cellulose I and cellulose II was achieved efficiently within one BC sample. The isotropic BC was converted to anisotropic material with unidirectional strip patterns because of the realignment of cellulose chains. The application of Digital Image

Correlation (DIC) was utilized in evaluating the strain distribution of patterned BC samples and the alteration of Poisson’s ratio between untreated BC, patterned BC and fully mercerized BC

VIII samples. The mercerized BC displayed controllable values of strength and toughness that showed its potential to be used in regenerative medicine and biomaterials manufacturing.

IX Acknowledgement

I would like to thank all of the people at EMAE department of Case western who made this work possible. My advisor, Dr. Ozan Akkus, always graciously gives me invaluable support, insightful suggestions, intelligent guidance and engagement. The door to Dr. Akkus office is always open whenever I have a question about my research or writing. I am also very thankful to

Dr. Clare Rimnac and Dr. Joseph Mansour for their valuable help and sound judgment. Thank you to Mousa Younesi for generously helping me immensely with everything microscopy, XRD, and

Raman related and answering my questions about unfamiliar techniques.

I am very grateful to my labmates, Hyungjin, Greg and Levent for rendering help and donating their time for discussions. It has been my great honor to work in such a lovely lab.

Finally I would like to thank my family for their selfless support, who have been at my side for all these years.

X Chapter 1 Background

1.1 Bacterial cellulose

1.1.1 History

Bacterial cellulose (BC) (Figure 1.1) is a naturally derived polymer produced by various species of bacteria, including Acetobacter, Rhizobium, Agrobacterium, Aerobacter,

Achromobacter, Azotobacter, Salmonella, Escherichia, and Sarcina [11].

Figure 1.1 Bacterial cellulose sample collected from our lab

11

The oldest known use of BC was as the raw material of nata-de-coco, a candy or dessert originated in Philippines. Nata-de-coco was a fermentation product of coconut water with a culture of Acetobacter xylinum, and the major component of nata-de-coco was BC [12].

BC has also been served as a diet beverage in Asia for many centuries, especially in form of the tea fungus production, Kombucha [13]. Kombucha is a product of the microbial activity of combing colonies of bacteria and yeast(Figure 1.2) [14]. The beverage claims several thousand years of history, and was originated in China around 220 BC (Roche, 1998). A few centuries later the drink was brought to Japan from Korea by the physician Kombu and was used for healing aliments [13]. At the beginning of the 20th century, the drink was traded into Russia and subsequently made its way to Eastern Europe in the early 1910’s [15]. Currently the beverage is gaining popularity in the West due to reported therapeutic benefits ranging from weight loss and anti-diabetic activity [16] to curing cancer and AIDS [17]. However, BC was first discovered in 1886 by A.J. Brown on the surface of a fermentative substance. Brown reported that under a favorable circumstance a white gelatinous membrane with the thickness of 25mm was generated by bacterium xylinum that was renamed as Acetobacter xylinum today.

Over the following one hundred years, because of its distinctive physiological and biological properties, BC has been widely used in multiple fields that range from the food industry and diaphragms of acoustic transducers to medical biomaterials and smart materials [18].

12 Figure 1.2 Symbiotic culture of bacteria and yeast

1.1.2 Synthesis and Chemistry of Bacterial cellulose

Alphaproteo-bacteria, Betaproteo-bacteria, Gammaproteo-bacteria and Gram-positive bacteria are the widely used type of bacteria that produce cellulose in today’s industry market

[19]. Members of the Enterobacteriaceae family isolated from the human gastrointestinal tract have also been reported having the ability to synthesize cellulose [20]. Regulators of cellulose synthesis have been determined in Escherichia coli and Salmonella [21]. Until now,

Acetobacter xylinum is thought to be the most recognized organism to produce cellulose.

Cellulose produced by Acetobacter xylinum owns remarkable properties such as high crystallinity, porosity, transparency, high tensile strength, and biocompatibility [22].

13

Formation of Uridine diphosphate glucose (UDPGlc) (Figure 1.3) is the precursor of

BC synthesis. The process is involved to transform glucose to glucose-6-phosphate (Glc-6-P), and then converting glucose-6-phosphate into glucose-1-phosphate (Glc-1-P) catalyzed by the cellulose synthase enzyme. Subsequently, every β-1-4 glucose molecule rotates 180 degrees with respect to the adjacent molecule, generating intermolecular hydrogen bonds, and polymerizes into single, linear β-1,4-glucan chains inside the bacterium body. Glucan chains are extruded out of cytoplasm membrane of bacteria through a row of macro pores along the long axis of the cell at the surface of the bacterium body. Glucan chains assemble together outside the cell envelope, aggregating into nanofibrils firstly with width in the nanometer range, followed by the further organization of small nanofibrils into long nanoribbons [23-27].

Figure 1.3 The synthesis process of BC.

14 UDP-Glc: Uridine disphosphoglucose; Gluc-6-p:Glucose-6-phospate; Fru-1-p: Fructose-1-phosphate;

Fru-6-p:Fructose-6-phospate; Fru-bi-p:Fructose-1,6-biphosphate; Fru-1-p:Fructose-1-phospate. ① : glucokinase ②: glucose-6-phosphate dehydrogenase ③: phosphoglucomutase ④: pyrophosphorylase uridine diphoosphoglucose ⑤: phophoglucoisomerase ⑥: fructokinase ⑦: fructose-1,6-bi-phosphate phosphatase ⑧: system of phosphotransferases ⑨: fructose-1-phosphate kinase.

During these processes a unique 3-D form network structure with highly uniaxially oriented nanofibrils and inter/intra molecular hydrogen bonds are formed. The interconnected

3D structure of cellulose is known for its high crystallinity, excellent water swelling ability and mechanical strength.

1.1.3 Structures of Bacterial cellulose

There are several crystal structures of cellulose (I, II, III, IV) (Figure 1.4.a). Cellulose type I and II are two forms of cellulose which can be found in nature [28]. Cellulose I is a ribbon-like polymer that glucan chains are arranged parallel to each other. Two polymorphs, cellulose Iα and Iβ, coexist in native cellulose though the ratio of Iα/Iβ is determined by the cellulose source [29]. Unlike cellulose I, in which chains are placed in a parallel orientation, cellulose II has an antiparallel structure (Figure 1.4.b). Cellulose II has been proved to be the most thermodynamically stable crystalline form and can be obtained by two approaches:

15 regeneration and mercerization [30]. Cellulose III is produced by liquid ammonia treatment of

cellulose I or II, and thermal treated cellulose III can consequently form cellulose IV. However,

the cellulose produced by bacteria is mostly cellulose I, which contributes to the superior

mechanical strength of BC (Young’s modulus of 15-20 GPa) [31].

Cellulose I Cellulose II

a b Figure 1.4 Cellulose types a. Four types of cellulose structure [2]. b. The supramolecular structure of cellulose I and cellulose II [3]

Plant cellulose has been extensively studied and intensively applied in paper and wood

industries. Plant cellulose fibrils construct an integral part of complex polysaccharide cell wall

matrix in contrast to the cellulose made by bacteria. Even though the plant cellulose has the

same molecular unit as that of bacteria, unlike BC, plant cellulose is not a pure form. Plant

cellulose requires radical purification pretreatment to separate it from the hemicelluloses and

lignin, which are tightly bound to cellulose [32]. Moreover, BC has superior properties over

plant cellulose such as higher tensile strength, higher degree of polymerization, thinner fibril

structure, better water swelling, higher porosity and larger surface area [33].

16 1.2 Culture conditions

Bacteria consume the carbon source of medium to create glucan chains with its high oxidative ability [34]. Besides the most established approach that uses sugars such as glucose, fructose, and sucrose as the carbon source for cultivation, some researchers focus on discovering a more economical and efficient carbon source in order to complement large-scale industrial production of BC [35]. In general, it is necessary to consider the following factors that could affect cellulose production: culture medium, culture conditions and byproducts.

Optimization of growth medium composition is essential for BC formation and furthermore improving the BC yield. The effect of carbon sources such as glucose, fructose and sucrose on

BC production has been evaluated. Besides carbon source, other nutrition factors such as nitrogen and ethanol together with the environmental conditions such as temperature and pH, also play important roles in the growth of BC [36]. Additionally, conventional culture methods are primarily under static environments, which request longer culture period and intensive manpower [9]. Because of that reason, bioreactors have attained great attention in recent years facilitate the synthesis of BC and lower the cost of BC production (Figure 1.5) [37].

17 a. Stirred Tank Bioreactor b. Airlift Bioreactor c. Rotary Disk Bioreactor

Figure 1.5 Different types of bioreactors designed to produce BC. Figure is from [9].

A relatively low acidic culture medium was desirable for bacterial cultivation.

Compared with the constant pH culture environment, culture medium with a slight natural shift toward acidic pH resulted in 1.5 folds higher BC yield [38]. It was also found that the optimal culture duration depends on bacterial strain type, ranging from 24h (k. xylinum BRC 2001) to

8 days (Acetobacter sp. V6)[39]. Furthermore, it was reported that in general the most favorable temperature for bacterial growth and BC production is in the range of 28 to 30 °C

[40].

1.3 Applications of BC in regenerative medicine

In recent years a growing body of work has focused on designing ideal biomedical devices from BC due to its advantageous features including the outstanding biocompatibility

18 and strong mechanical strength. Although there is no work proving BC is degradable in human body yet, BC has been evaluated to have great potential as a biomedical material such as wound dressing, artificial blood vessels, artificial urethra, bone tissue scaffold, artificial cartilage, drug delivery template, artificial knee menisci and dental treatment material. Among all these biomedical applications, wound healing dressing has attracted most interest and it has developed as a FDA (Food and Drug Administration) approved commercial biomaterial (Bios

King biocellulose film). Taking advantage of the high moldability, tubular BC was characterized as artificial vessels inspired by the work of Klemm et al. in 2001 [41]. Porous BC loaded with certain medicine can serve as a drug delivery vehicle [42]. Moreover, BC is very attractive as a scaffold for tissue engineering and many studies have reported its good tissue integration both and in vivo [43-46].

1.3.1 Wound dressing

Wound dressings for treatment of burns and chronic wound are crucial in medical and pharmaceutical market for centuries. Historically, the primary function of traditional dressings such as natural or synthetic bandages, cotton wool, lint, gauzes, woven and non-woven sponges as well as tulle dressings is to keep the wound in a dry environment, so as to absorb exudate and prevent the bacterial infection [47]. However, over the past few decades, it has been proven that a warm moist wound environment optimizes the wound healing process

19 significantly. Modern dressing materials have been developed to aim at improving the wound healing as well. Depending on the wound type, the phase of wound healing and patient conditions, dressings should have following desirable characteristics: provide a warm moist environment; permit oxygen circulations; remove excess exudates; form a tight physical barrier between the wound and the surrounding environment; prevent bacterial invasion (Figure

1.6)[48].

BC is widely used as a wound dressing material due to its unique physical and chemical attributes that fulfill all essential requirements for a dressing material. Solway [49] introduced that the application of BC to a diabetic ulcer enhanced the rate of wound healing and shortened the time course of epithelization. The greater absorptive capacity of BC membranes allowed for the dissipation of exudate, and trapped platelets, which reduced pain and acted as a regenerative tissue scaffold. BC created a close contact to the patient’s skin and facilitated the healing process compared with the traditional gauze. Importantly, the transparency feature of

BC allowed for continuous clinical observation of the healing progress [50].

Figure 1.6 BC applied on a wound located on the hand as a dressing material. Figure is from [6] .

20 BC can be modified to promote the wound healing process. One of main problems of

BC is BC itself has no antimicrobial activity against bacterial invention [51]. Several approaches were applied to successfully introduce antimicrobial properties into BC materials and enhance its effectiveness as a treatment for highly contaminated wound. Silver nanoparticles have been incorporated into dressing material for more than a century because of its remarkable ability to kill bacteria. In general, silver nanoparticles can be impregnated by direct diffusion of previously synthesized BC [48]. Helenius et al. [52]evaluated the in vivo biocompatibility, implanting BC in rats for 1, 4 and 12 weeks. No signs of chronic inflammation around the implanted BC or in the incision were observed at any time point, and

BC implants did not induce any foreign body reaction. Additionally, BC was very well integrated into the host tissue in this study, and after 12 weeks the fibroblasts were utterly integrated into the BC structure and had synthesized new collagen. Pertile et al. [53] modified the BC membrane with the nitrogen plasma in a plasma reactor. In this research, the modified

BC had better cell adhesion and biocompatibility resulted from the increased roughness and porosity.

1.3.2 Artificial blood vessel

Cardiovascular disease (CVDs) is the leading cause of mortality for both men and women in western countries. 17.5 million people die each year from CVDs, an estimated 31%

21 of all deaths worldwide. Autologous vascular grafts remain the most common treatment.

However, synthetic biomaterials have been extensively studied as substitutes for many patients

[54].

Figure 1.7 BC tubes with different inner diameters 1.5 mm, 2.4 mm, 3.0 mm, 4.0 mm, and 6.0 mm. Figure is from [5].

Many strategies have been deployed to improve the compatibility and effectiveness of vascular grafts. Among all the renewable and natural biomaterials, BC has been explored as an artificial blood vessel in recent years due to its unique properties (Figure 1.7). Fink et al. [55] verified that BC material did not induce plasma coagulation to any great extent, and generated the least and the slowest activation of the coagulation cascade in comparison with existing commercially available graft materials of poly(ethyleneterephtalate) (PET) and expanded poly(tetrafluoroethylene) (ePTFE). It is also important to evaluate mechanical properties for material used for vascular grafts applications. A pellicle of BC that contains 99% water is not as strong or elastic as an entire blood vessel that contains less water(70%-80% ) [56].

Schumann et al. [57] conducted in vivo experiments in rats and pigs with the purpose of proving that BASYC® could be applied in tissue-engineered blood vessels as part of programs

22 in cardiovascular surgery. Rats were allowed to grow for 1 year after the BASYC® was attached in an artificial defect of the carotid artery. These long-term results showed the ingrowth of active fibroblasts and the integration on BASYC®. Scherner et al. [58] analyzed the in vivo performance of BC grafts in 10 sheep after implantation of BC grafts with a length of 100 mm for a period of 3 months. They revealed a patency rate of 50% and also observed a neo-formation of a vascular wall-like structure along the BC scaffold, indicating that BC grafts could be potential scaffolds for small-diameter tissue-engineered blood vessels.

Figure 1.8 Small caliber BC graft in vivo. Figure is from [7]

1.3.3 Drug delivery

There has been an increasing interest in the use of natural materials as drug delivery vehicles. BC has been used in a number of studies to fabricate drug delivery systems. BC could

23 interact with drug nanoparticles, absorb drug nanoparticles and release the loaded drug controllably [59].

BC membranes were tested as supports for drug topical delivery owing to its good skin tolerance in vivo patch test [60]. Molecularly imprinted polymer (MIP) and BC membranes were integrated to form composite membranes that acted as a transdermal delivery system of the S-enantiomer of propranolol, an antihypertensive drug. The cellulose membranes containing modified pores and a surface with imprinted polymer could selectively convey

S-propranolol through the MIP composite [61]. Homogeneous caffeine-loaded BC membranes presented significantly lower permeation rates than those obtained with traditional formulations.

The addition of glycerol would increase the flexibility of the caffeine-loaded BC membranes and result in a doubled swelling capacity compared with the pure BC membranes [62]. A drug loading process in BC membranes was developed for lidocaine hydrochloride and ibuprofen.

The systematic in vitro diffusion study with Franz cells reported that the integration of lidocaine hydrochloride in BC membranes provided lower permeation rates than those obtained with the conventional formulations. However, the permeation rate of ibuprofen in BC membrane was three times higher than that of a PEG400 solution, revealing that BC was able to provide an alternative permeation rate for specific drugs [63]. Depending on the cultivation process and post modifications, it is possible to optimize the surface structure and biochemical properties of BC membranes for a controllable drug delivery system.

24 1. 3. 4 Bone tissue engineering

Bone tissue engineering is an alternative strategy to develop a 3D scaffold to either prompt formation of bone from the surrounding tissue or to act as a carrier or template for implanted bone cells or other agents. Over the last decade, development of biomaterials used for making porous scaffold has been an intensive area of research [64].

Figure 1.9 In vivo operation of BC membrane as a barrier membrane on guided bone regeneration. Figure is from [1].

An ideal premade porous scaffold for bone tissue engineering should possess interconnected porous structure and thus the organized structure might guide cells to grow at various stages of development. A variety of biomaterials have been evaluated and these biomaterials can be classified into natural and synthetic biomaterials. Naturally derived BC can serve as a matrix to support cell growth. BC can be easily modified and altered by controlling the fermentation procedure and introducing various functional nanoparticles to it [65, 66]. In

25 attempt to use BC as cellular scaffold for guiding tissue repair and bone regeneration, it is essential to evaluate tissue reactions to a BC membrane after implantation. BC was implanted into lumbar subcutaneous tissue of 25 mice. Foreign body reaction was not observed at any time point through the study period while a mild inflammatory reaction was observed at day 30

[67]. In another study, second harmonic generation (SHG) microscopy was used to visualize the development of collagen on BC scaffold seeded with osteoprogenitor cells. BC permitted an immediate collagen synthesis during the first days of growth. Moreover, it was monitored that the osteoprogenitor cells were able to produce collagen inside compact regions of cells in the

BC micropores [68]. Additionally, the incorporation BC to antibiotic bone cement would improve antibiotic release by 1.3-fold as compared with traditional cement. Compression strength of the bone cement was also significantly greater from the BC impregnated antibiotic cement (92.1±4.8MPa) than from the traditional antibiotic cement (75.3±8.1MPa) [69].

In bone tissue engineering, the appropriate pore size of material is dependent on the specific size of cells. However, the pore size on the surface of BC is quite small (less than

100-300 nm) and the pore arrangement on the cross-section of BC is irregular. A combined approach consisting of acetic acid treatment and freeze-drying operation was applied to enhance the porosity form 50.3% to 76.43%, which led to a better cell viability for human fibroblast cells on BC membranes [70]. An innovative BC sponge with high porosity of

90.42±0.25% and high surface area of 92.81±2.02m2/g was obtained with the emulsion freeze-drying method [71]. Another study proposed the use of irreversible electroporation as a

26 novel biofabrication method to create appropriate porosity in the scaffold for orthopedic applications by varying certain parameters such as the voltage applied, number of pulses, frequency and duration between treatments [72]. Microporous BC scaffolds with an interconnected network of pores in size 300-500 μm were successfully obtained, using paraffin wax microspheres as the porogens during the fermentation process. In vitro biomaterial-cell constructs were evaluated by culturing MC3T3-E1 osteoprogenitor cells on the microporous

BC scaffolds. Cells were found to occupy pores in the top two thirds of the microporous BC scaffolds and cells appeared to be more compacted within the pores [8].

Figure 1.10 The morphology of porous bacterial cellulose produced with paraffin wax particles of size 300–500 µm. Figure is from[8].

1. 3. 5 Cartilage

It is well established that the damaged articular cartilage has a very limited potential for healing, resulting in a loss of joint function [73]. To overcome this low regeneration capacity of

27 cartilage, the use of scaffolds in tissue engineering of cartilage is rather essential to support new growing tissue [74]. In cartilage tissue engineering, the physical and biochemical properties such as 3D network structure, cell adhesion and proliferation are crucial for the scaffolds on cartilage repair process [75]. Over the past decade, BC has become an attractive scaffold material for cartilage tissue engineering because of its superb biocompatibility. Micro-channel

BC seeded with functional cells was confirmed to have potential as a meniscal scaffold with its favorable cells guidance ability [76]. Furthermore, the small sizes of the pores in BC surface might limit the ingrowth of cells into BC scaffolds, particularly the dense top layer of the material. Laser-patterning technique was applied to produce 3D perforated BC with the attempt to induce structural modifications and thus allow connective tissue cells seed into BC [77]. BC network of pores with diameters ranging from 300 to 500μm was prepared by utilizing agarose microparticles during fermentations to control the pores size [78].

1. 3. 6 Dentistry

Given that the sterilization of dental root canal requires a treatment material with high absorbency, excellent biocompatibility and the ability to improve intracranial medication, Dried

BC sheets were pressed, and were subsequently rolled into a point form according to ISO 45 standard. In all three solutions including saline, K+ free electrolyte fluid and electrolyte fluid, the absorption rate of BC was 85-fold its weight while the absorption rate of commercially

28 available plant points (PP) was about 8-fold its own weight. In addition, BC showed noticeably higher expansion (3-fold original thickness) in comparison with PP (no expansion) during soaking period. In in vivo biocompatibility experiment conducted on rats, BC exhibited less inflammatory cell as compared with PP 2 months after implantation. Moreover, BC could hold a greater amount of liquid medicament, and the release of trypan blue was significantly higher than PP in the drug release test [79]. This finding strongly supported that BC possessed many favorable characteristics for the use of a dental root canal treatment material.

1. 3. 7 Neurosurgical Applications

Neural tissue repair and regeneration has received a good many attention and polymeric biomaterials are widely preferred as scaffolds for nerve regeneration. Natural polymers including chitosan, gelatin, collagen and alginate have been studied in various nerve regeneration approaches (Figure 1.11) [80]. In recent years, BC has also emerged as a promising biomaterial for its application as a scaffold in neuronal tissue engineering.

Tubulization, which makes an implant in the form of cylinder, has been considered to be the most promising method to repair damaged peripheral nerves. Taking advantage of the prominent biocompatibility, BC was found to be an ideal material for the reconstruction of damaged peripheral nerves. Kowalska-Ludwicka et al. [81] developed BC guidance channels and analyzed its in vivo regenerative effectiveness in femoral nerve of Wistar rats. The

29 histological outcome confirmed that the application of BC tubes successfully lowered the formation of neuroma and the excessive proliferation of connective tissue (35%) was quite lower than that of control group (86.67%). The effect of BC as a new substitute for dura was tested in rabbit model with dural defects, pointing out that BC membranes could evenly cover the surface of brain without adhesion. In order to prevent cerebrospinal fluid leakage, one of the most serious complications in cranial and spinal surgery, no adhesion between dura and brain tissue was a desirable criterion for artificial dura mater [10].

Figure 1.11 Dura defects were patched by BC on the left side. Figure is from [10].

1.4 Mercerization

One of the processes by which cellulose II can be obtained from cellulose I is mercerization. Unlike cellulose I, in which chains are placed in a parallel orientation, cellulose

II has an antiparallel structure [82]. Mercerization is an alkaline treatment process generally by

30 using NaOH that induces natural fibers to undergo crystalline structure changes. The conversion from cellulose I to cellulose II by mercerization is irreversible. Previous findings elucidated the effects of mercerization treatment on physical properties of various plant based cellulose (cotton, sisal, jute, bamboo, flax, curaua or coir) [83-89]. Mercerization improves the purity of plant cellulose by removing substances such as hemicellulose, lignin and pectin in the primary cell walls of plant [90]. Additionally, mercerized plant cellulose exhibits higher wear resistance [83] and higher thermal stability [85] than native plant cellulose. Revol et al.

[91]reported that in high crystallinity cellulose (such as valonia and ramie), mercerization reduces crystallinity and crystallite size while for low crystallinity cellulose (such as celery and swiss chard) crystallinity and crystallite size increased upon mercerization.

1.5 Conclusion and perspective

Bacteria consume the carbon source in the medium initially and utilize the carbon source to create sufficient glucan chains to form the complete BC on the surface between the air and culture medium. The reason of why bacteria create cellulose is still not fully understood whereas some scholars postulate cellulose as an adherent environment that provides protection.

A wide variety of carbon sources have been tested so far and fructose has been considered to be the most favorable substrate among conventional carbon sources including fructose, sucrose and glucose. The addition of ethanol has been proved to stimulate the BC yield. As one of essential elements that allow life to exist, nitrogen addition also prompts the BC yield to some

31 degree. Although some other chemicals have been confirmed to enhance BC production, the developments of economical and industrially valuable culture medium still deserve more attention. An aeration and agitation culture process is more commercial for industrial production as compared with static environment.

Generally, alternation of BC structure and incorporation of secondary components into

BC are two mainly used approaches to modify the properties of BC. Recent findings have fueled the development of BC as a promising bioengineering material. As of now, a reliable synthesis system of BC formation has been gradually established. Numerous researches have shown the great potential of BC as a biomaterial. Taking advantage of the biocompatibility of natural biomaterials, many studies tried to combine BC with other broadly used biomaterials such as collagen, chitosan, gelatin and alginate by immersion or mixture approaches, and therefore obtained quite admirable results.

Table 1.1 Mechanical properties of BC based composites

Composite Young’s Modulus Ultimate tensile strength (MPa) Strain at Reference

（GPa） break (%)

Pure BC 0.9-6.4 95.1-200 6.5-11.8 [92]

BC/Collagen 0.63-9.5 76.7-275 4-14.5 [92, 93]

BC/Gelatin 3.9 114 4 [94]

BC/Chitosan 0.782-10.29 54-132.19 2.8-28.54 [95-97]

BC/ hyaluronan 0.47-0.61 0.91-1.03 38.39-48.14 [98]

32 1.6 Motivation of this thesis

Unlike plant cellulose, the mechanical properties of BC need to be elucidated. BC is strong but invariably brittle, therefore we need to find a way to provide BC high ductility.

Studies to date have mercerized cellulose fully and the effects of partial mercerization on mechanical properties have not been investigated yet. Furthermore, unlike the plant cellulose, the effect of mercerization on the mechanical properties of BC remains unknown. In this study, controlled mercerization is employed as means to tune the mechanical properties of BC. Taking advantage of BC’s outstanding biocompatibility, present work evaluates the potential of BC in various bioengineering applications which require stiff materials (such as bone) to compliant

(such as skin). The results of the study will demonstrate that such disparate mechanical properties can be attained by controlled mercerization of BC.

An innovative approach with the use of a high-resolution commercial laser printer was developed to apply patterns on BC samples with a high degree of accuracy during the mercerization treatment. Patterns were designed to protect certain parts of the material from mercerization while the rest of material was subject to the alkaline treatment. As a result, a pre-designed combination of cellulose I and cellulose II was achieved efficiently in BC samples.

First, it was possible to change the isotropic BC to anisotropic material with unidirectional strip patterns because of the realignment of cellulose chains. Different values of Young's modulus

33 were obtained in two orthogonal testing directions. In general, the unmercerized BC domains gave rise to the global anisotropic behavior. Additionally, a BC sample with mercerized

re-entrant hexagonal honeycombs patterns with stiffer BCI frame structure and ductile interior

region of BCII was achieved. We demonstrated the application of Digital Image Correlation

(DIC) in evaluating the strain distribution of patterned BC samples and the alteration of

Poisson’s ratio between untreated BC, patterned BC and fully mercerized BC samples.

34 Chapter 2 Experimental

2. 1 Materials

2. 1. 1 Raw materials and processing

Probiotic kombucha beverages were purchased from local market. Kombucha scoby consists of bacterial and yeast species, predominantly, Gluconacetobacter, Acetobacter, and

Zygosaccharomyces. Kombucha scoby was grown in a 5 % (w/v) glucose medium under the static environment at room temperature for 14 days.

2. 1. 2 Removal of bacteria

Cellulose pellicles were immersed in 2% aqueous solution of NaOH at 70°C for 3 hours and were repeatedly washed three times with deionized water to remove bacterial debris. After this treatment, samples were air-dried at room temperature and stored at 4 °C for later tests.

35 2. 2 Sample preparation

2. 2. 1 Mercerization

Dried BC samples were incubated in NaOH solutions at concentrations of (0 M, 3 M, 4

M, 5 M and 7 M) for 15 minutes at room temperature. After the treatment, BC samples were rinsed in deionized water until the attainment of a pH value of 7.

2. 2. 2 Preparation of patterned BC samples

A laser printer (Ecosys m3540idn, 1200 dpi) was used to print the pre-designed patterns on dried BC sheet samples. Designed patterns were printed on one side of the BC and the other side was masked by full black print (Figure 2.1).

Figure 2.1 The patterning process: designed patterns are printed on one side of the dried BC

sheet, and black color is printed on the whole backside. Patterned BC is treated with 7 M NaOH solution at room temperature for 5 minutes. After the treatment, BC sample is thoroughly washed with deionized water to remove NaOH remaining and the toner.

36

Figure 2.2 (a). Geometric parameters of striped patterns. (b). Rectangular samples are cut from the striped patterned BC sheet along two different orientations. (b.1). Patterns are aligned in longitudinal direction; (b.2). patterns are aligned in transverse direction. Scale bar is 1.5 mm

Striped patterns on BC samples with the dimension of 0.6 mm × 3.6 mm and the spacing of 0.4 mm were prepared to evaluate the effect of unidirectional patterns on the mechanical properties of the BC samples and how the anisotropic pattern can provide us with anisotropic mechanical properties (Figure 2.2).

Re-entrant hexagonal honeycombs patterns on BC samples were produced to analyze the effect of this pattern on Poisson’s ratio of an auxetic structure made by BC samples (Figure

2.3). Patterned BC samples were incubated in 7 M NaOH at room temperature for 5 minutes.

Samples were then rinsed in copious amount of deionized water repeatedly until the attainment

37 of a pH value of 7.0 for water that used for rinsing. Resulting samples had fine patterns since the printer toner protected masked parts of the BC samples from the mercerization. Control groups were unmercerized BC samples and BC samples that were fully mercerized with 7 M

NaOH solution.

Figure 2.3 (a). Geometric parameters of the re-entrant hexagonal honeycombs structure; (b).

Picture of BC sample with re-entrant honeycombs patterns. Scale bar is 3.5 mm.

Figure 2.4 Re-entrant honeycomb structure expends under stretching.

38 2.3 Shrinkage test

Dried BC sheets were punched into circular samples with diameters of 10 mm (Figure

2.5). Samples were soaked into different concentrations of NaOH solutions (0 M, 1.5 M, 1.75M,

2 M, 2.125 M, 2.25 M, 5 M, 7 M and 10 M) for 30 minutes at room temperature.

In order to evaluate the effects of different monovalent cations on the shrinkage, BC samples were treated in 3 M KOH solution and 3 M NaOH solution for 18 min at room temperature respectively.

Videos were recorded to track the dimensional changes of each sample temporally during the process of the mercerization. Images were collected at every 20 seconds at the first 4 minutes, every 30 seconds from 4 minutes to 10 minutes and the time interval was increased to

120 seconds till the end of the recording. The change of the diameter was measured by using

Image J software. Shrinkage rate was calculated by taking the derivative of the shrinkage versus time functions before BC samples obtained the steady state. Shrinkage rate in Table 3.1 was determined when reaction time was 60s.

Figure 2.5 Unmercerized (a.) and mercerized (b.) samples. Mercerization was

performed in 7M NaOH for 30 min at room temperature. 39 2. 4 Mechanical testing

Tensile testing of BC samples was carried out using a universal testing machine equipped with a 5 lbs load cell (Testresources, Shakopee, MN). Rectangular strips were cut off from a BC sheet. To compare the mechanical behavior when measured in different directions and investigate the anisotropic mechanical behavior of samples, rectangular samples were cut from the striped patterned mercerized BC sheet along two different orientations (y direction and x direction as shown in figure 2.2). All samples were hydrated in deionized water for 1 h prior to testing. The width and thickness of samples were measured by digital caliper and micrometer respectively. Specimens were tested to failure at a displacement rate of 0.167 mm/s.

Ultimate tensile strength (UTS) and elongation at break were defined as the maximum stress and strain that the sample could withstand before fracture, respectively. The slope of the curve in the linear elastic region was calculated to obtain Young’s modulus (E). Toughness was calculated by numerical integration of stress-strain curves.

2. 5 Digital Image Correlation (DIC) set up and analysis

BC strips for DIC tests were dipped in 7 M NaOH solution in a way to create alternating mercerized and unmercerized stripes along the length of strips. Half mercerized/ unmercerized BC samples and BC samples with re-entrant hexagonal honeycombs patterns were coated with a thin layer of white paint initially to create contrast for imaging after which

40 fine black speckle patterns were applied using an air-brush (Master Airbrush, G233) (Figure

2.6).

Figure 2.6 The sample prepared for the DIC analysis. Scale bar is 1mm.

Mechanical tests were performed in tension as described previously at a displacement rate of 0.0167 mm/s. Digital image correlation was conducted in 2D using a single camera and images were captured using Vic-Snap 8 (Correlated Solutions, Inc., Columbia, SC) at every

250 ms until fracture. A 3 mm pitch grid (Correlated Solutions, Inc., Columbia, SC) was employed to calibrate the scale. The strain distribution was calculated using Vic-2D (Correlated

Solutions, Inc., Columbia, SC).

41

2.6 Measurement of global Poisson’s ratio

In order to estimate strains in the transverse direction and the axial direction individually, pre-marked lines were selected to measure the strain in both x and y directions during the elastic deformation (Figure 2.7). Pre-marked lines stick to the sample and move with the sample during the tests. For each group, the altered lengths of the pre-marked lines were calculated to analyze the strain and the Poisson’s ratio was determined by formula (2)

! !"#$%&'"%' ν = − (2) ! !"#!$

Figure 2.7 Strain was measured by calculating the distance changes of the pre-marked lines. Scale bar is 1.5 mm.

42 2. 7 Raman spectroscopy

Mercerized and unmercerized samples were analyzed via Raman spectroscopy using a

785 nm Raman system (Horiba Jobin Yvon, Edison, NJ, USA). The spectrum was obtained from 3 different locations on the samples, and each spectrum was obtained as the average of 30 consecutive spectra collected for 10 s each. Background subtraction from the spectra was performed using Labspec software (Horiba Jobin Yvon). Data were utilized to investigate the effect of mercerization on molecular and crystalline structure of the cellulose samples.

2. 8 X-ray Diffraction

XRD patterns for BC and mercerized BC were recorded on Bruker Discover D8 X-ray diffractometer using CoKα radiation generated at 45 kV and 40 mA. Scans were obtained from

10 to 40 degrees in 0.05 degrees per second with a step size of 0.025°. Crystallinity index was

calculated from the ratio of the height of the 002 peak (I002) and the height of minimum (Iam)

between the 002 and the 110 peaks (Iam) as given by formula (3).

(!!!"!!!") ��� = ×100 (3) !!!"

where I002 is the intensity of the peak at 2θ = 26.2° for unmercerized BC and 2θ = 25.8° for

mercerized BC. Iam is corresponded to the amorphous content at 2θ = 21.2° and 20.2° for unmercerized BC and mercerized BC respectively.

43 2. 9 Statistical analysis

Data are reported as mean ± standard deviation (SD). Data analysis was performed by one-way ANOVA followed by Tukey’s pairwise comparison to determine significant differences in shrinkage and mechanical properties of the samples. Statistical significance was set at P<0.05.

44 Chapter 3 Results

3.1 Effect of NaOH concentration on BC shrinkage

Dimensional changes as a result of mercerization involved a rapid shrinkage phase whose rate declined over time toward a steady state dimension (Figure 3.1). The extent of shrinkage was dramatic (Figure 2.5). There was a threshold of 1.75 M NaOH concentration above which shrinkage took place (Figure 3.2). When NaOH concentration increased to 2 M, a

12 ± 1.2% shrinkage in the diameter occurred at the steady state. Increasing the concentration of NaOH increased the rate and degree of mercerization, and diametric shrinkage reached the maximum of 47 ± 1.6% at 10 M NaOH. There is a bilinear relationship between the concentration of NaOH solution and the final shrinkage of diameter. Rapid increase in the diameter shrinkage was observed in the NaOH concentrations, ranging from 1.75 M to 2.25 M. while increasing the concentration over 3 M led to a smaller amount of increase in the resulting shrinkages.

45 Figure 3.1 Shrinkage of BC after reacting with different concentrations of NaOH solution at room temperature (n=5/group). Bars indicate the standard deviation.

Figure 3.2 A rapid increase in % shrinkage at the steady state was observed at NaOH concentrations between 1.75 M and 2.25 M, followed by a lower amount of increase at higher concentrations. Bars indicate standard deviations (n=5/group).

46

No significant difference was observed in the rate of mercerization between BC discs treated with 3 M KOH (0.28 %/s ± 0.024 %/s ) vs. 3 M NaOH (0.298 %/s ± 0.012 %/s) (P >

0.05) (Figure 3.3). However, the resulting shrinkage of NaOH mercerized BC (26.63 ± 1.4%) was significantly higher than that of BC mercerized with KOH solution of the same concentration (21.4 ± 1.6%) (P = 0.002).

Figure 3.3 The shrinkage of bacterial cellulose treated with 3 M KOH and 3 M NaOH for 20 min under room temperature (n=5).

47

3.2 2D strain distribution of mercerized BC

Strains along the direction of tensile loading (Eyy) varied between mercerized regions

(0.0615 ± 0.0141) and unmercerized regions (0.0158 ± 0.0087). A stress concentration effect was observed at the junction between mercerized higher and lower modulus zones, resulting in strain values of (0.0878 ± 0.0156) (Figure 3.4).

Figure 3.4 Strain map of Eyy along the tensile loading direction demonstrates alternating high and low level strains corresponding to mercerized and unmercerized strips. Elevated strains were present at the interface between hard and soft phases, indicating the presence of a stress concentration. Scale bar is 1 mm.

48 3.3 Effect of NaOH concentration on mechanical properties

Typical stress-strain behavior of mercerized and unmercerized BC involved a linear relationship followed by an abrupt failure with little or no plastic deformation taking place

(Figure 3.5). Substantial changes were observed in the slope and failure properties of stress-stain curves as a result of controlled mercerization. Untreated BC was the stiffest and strongest of all groups. Stiffness and strength decreased with increasing NaOH concentration along with an increase in failure strain (Figure 3.6). Ultimate tensile strength of 7 M NaOH treated BC samples was 64% of the strength of unmercerized BC (Figure 3.6 c). In a similar fashion to tensile strength of BC, the Young’s modulus of BC reduced 30-fold (P< 0.001) from

10 GPa to 0.3 GPa with increasing concentration of NaOH (Figure 3.6 a). The strain at failure of BC samples treated with 7 M NaOH (50.8 ± 5.7%) was 10-fold greater than that of unmercerized BC samples (P< 0.001)(Figure 3.6 b). While increasing NaOH concentration decreased strength and Young’s modulus of BC, the toughness of BC showed an increase till 4

M NaOH (64 ± 15 MJ/m3). The toughness did not change significantly (P > 0.05)at concentrations greater than 4 M (Figure 3.6 d).

49 Figure 3.5 Effect of NaOH concentration on mechanical behavior of BC (n=7). Typical

stress-strain curves of native BC and BC treated with different concentration of NaOH.

Figure 3.6 a. Effect of NaOH concentration on Young’s modulus of BC (n=7, significant difference were found among all groups P<0.05). b. The elongation at break increased about 10-fold when BC was mercerized at higher concentration of NaOH. (n=7, significant difference were found among all groups P<0.05). c. Ultimate strength did not differ significantly between untreated BC and 3 M NaOH mercerized BC. (n=7, two significantly different groups are connected by lines P<0.05).d. The toughness of BC reached a steady state value by 4 M NaOH. (n=7, two significantly different groups are connected by lines P<0.05).

50 Table 3.1. Mechanical Properties of Mercerized BC (n=7)

NaOH Diameter Shrinkage Young’s Ultimate Elongation at Toughness

(Molarity) shrinkage rate Modulus (GPa) Tensile break (%) (MJŸm-3)

(%) (%/s) Strength

(MPa)

0 0 0 10.3±1.7 444±115 5.4±1.6 15.13±7.7

3 26.6±1.4 0.17±0.02 2.1±3.8 355±65 15.4±3.0 31.2±10.5

4 * * 1.0±0.1 339±54 33.9±3.7 64.0±15.8

5 34.4±0.6 0.24±0.003 0.6±0.1 248±55 38.7±1.5 51.6±13.5

7 42.2±2.2 0.32±0.02 0.3±0.05 168±31 50.8±5.7 45.2±12.0

* Shrinkage of 4 M NaOH treated samples were not tested.

Figure 3.7 Mechanical behavior of BC samples mercerized under different temperatures (n=5). No significant difference (P<0.05) was observed between each group.

51 3.4 Raman Spectra and XRD

Polymorphic changes of cellulose I and cellulose II were evaluated by Raman spectroscopy (Figure 3.9). As it can be seen, a group of weak bands at ~ 900 cm-1 which are related to H-C-H and H-C=O bending were observed in both mercerized and unmercerized BC

[99]. In the 900 ～ 950 cm-1 region, peaks of unmercerized BC and mercerized BC are different in intensities and shape. In the 950 ～ 1180 cm-1 region, several peaks with high intensities were observed in both unmercerized and mercerized BC. The potential energy distribution is dominated by C-C and C=O stretching motion and small amounts of H-C-C,

H-C=O and skeletal atom bending. For unmercerized BC, intensity of 1095 cm-1 band, attributed to C-O-C glycoside stretching motions, was 1.5 times higher than that of mercerized

BC. The cystallinity of BC and mercerized BC were found to be 90.1 % and 60.5 % respectively from XRD analysis (Figure 3.8).

52 Figure 3.8 Typical XRD spectra for untreated BC (BCI) and mercerized BC (BCII) films.

Figure 3.9 Typical Raman spectra for untreated BC (BCI) and mercerized BC (BCII) films.

53 3.5 Effect of mercerized patterns on the mechanical properties of BC

Striped patterns were applied onto BC to evaluate the effect of mercerized pattern on the mechanical properties of BC. Two groups were cut from one large BC sheet (Figure 2.2).

One group of BC cut out samples had stripes aligned in the direction of the loading axis, and the other group had striped patterns aligned at a right angle (i.e. transverse) to the loading axis.

Striped patterns have significantly changed Young's modulus of samples. The Young's modulus along the long axis of longitudinally patterned samples (281.3 ± 80.3 MPa) was significantly larger than the modulus (144 ± 20.5 MPa) of the transversely patterned samples (Figure 3.10 a)

(P = 0.02). At the same time, failure strain (13.78 ± 3.29%) of samples with mercerized pattern in y direction were significantly lower than failure strain of samples patterned along the x axis

(25.71 ± 8.07%) (Figure 3.10 b) (P = 0.01). No significant difference was observed in the UTS of these two groups of samples (Figure 3.10 c) (P > 0.05). In comparison with the fully mercerized samples, untreated BC demonstrated significantly higher stiffness (P < 0.001) and strength (P = 0.01) (Young’s modulus of 776 ± 135.3 MPa and the UTS of 75.9 ± 15.6 MPa).

Fully mercerized BC exhibited lowest Young’s modulus (114 ± 12.26 MPa) and UTS (19.1 ±

3.7 MPa) before fracture as compared with patterned BC samples and untreated BC samples.

54

a. b.

at break (%) break at

c. d.

Stress (MPa) Stress

Figure 3.10 The effect of striped patterns on mechanical properties of BC samples. (n=5, significantly different groups are connected by lines, P<0.05). a. Striped patterns affect the Young’s modulus of mercerized BC. b. There was a significant difference in the strain values of mercerized BC with patterns in transverse direction and mercerized BC whose patterns are in axial direction under tensile test. c. Ultimate strength did not differ significantly between the mercerized BC with striped patterns when tested in two orthogonal directions. d. Representative stress-strain curves of untreated BC, different direction patterned BC and fully mercerized BC.

55

3. 6 Effect of mercerized patterns on the Poisson’s ratio

Strain maps of transverse strain (Exx) and axial strain (Eyy) obtained from DIC analysis were shown in Figure 5. Several measurement points were selected over three units to further

assess the pattern dependence of strains distribution. In Eyy map (Figure 3.12), it was clearly

visible that the localized strain distribution was in good agreement with the pattern. Stress concentration was present over the regions transitioning between unmercerized and mercerized

areas under the tensile load. Eyy varied in magnitude between unmercerized and mercerized regions (Figure 3.12). The Exx demonstrated an oscillating variation in values from positive to negative corresponding to the patterns (Figure 3.11). In the transverse direction, tension and compression were observed in the specimen under the axial force (Figure 3.11). The Poisson’s ratio of mercerized BC (0.45 ± 0.05) was significantly higher than that of unmercerized BC

(0.32 ± 0.04) (P < 0.001), while the Poisson’s ratio of re-entrant hexagonal honeycombs patterned BC decreased to 0.29 ± 0.03 (Figure 3.13).

56 Figure 3.11 a. Exx strain map of a BC sample with re-entrant hexagonal honeycombs patterns. Scale bar is 3.5 mm b. Exx strain map of magnified region. c. The Exx strain of points along the x axis. Scale bar is 3 mm.

Figure 3.12 a. Eyy strain map of a BC sample with re-entrant hexagonal honeycombs patterns. b. Eyy strain map of magnified region. c. The Eyy strain of points along the y

axis. Scale bar is 3 mm.

57 Figure 3.13 Comparison of Poisson’s ratio values. Patterned BC had a significantly lower value of Poisson's ratio compared with fully mercerized BC. (n=3, two significantly different groups are connected by lines, P<0.05).

58 Chapter 4 Discussion and conclusions

Mercerization is a well-established process to enhance the affinity of cotton fibers for dyes in today’s textile industry [100]. It has also been demonstrated that mercerization can modify the structure of BC [4]; thus, this study utilized mercerization to tune mechanical properties of BC to render it suitable for a broad range of applications. Our findings indicated that we were able to tune the stiffness of BC from a level that is similar to that of bone to a level that is similar to tendon.

The mechanism of mercerization has been studied extensively [101, 102]. At the first stage, Na-cellulose is formed at room temperature, involving the disruption of hydrogen bonds and the swelling of cellulose crystallite [103]. The type of alkali cellulose (Na-cellulose I or

Na-cellulose II) that is formed depends on the alkali concentration, temperature and subsequent treatments [104]. The penetration of concentrated NaOH solution within native crystals increases the lateral spacing between parallel cellulose chains and results in the expansion of microfibrils and breakage of some of the hydrogen bonds which are replaced by sodium atoms.

Swollen microfibrils relax and organize in a coiled conformation (Figure 4.2). Based on this new allomorph of cellulose molecules, chains of opposite polarity become more available and form an antiparallel arrangement for stacking of the hydrophobic plane [31, 105]. SEM figures revealed the surface texture difference between native BC and mercerized BC (Figure 4.1).

59 Mercerized BC exhibits a more compacted structure due to the shrinkage.

Figure 4.1 Scanning electron microscope (SEM) figures of native BC (a) and mercerized BC (b). Scale bar is 1 µm.

Figure 4.2 The mechanism of mercerization on BC. Figure is from [4]

Raman spectra have further demonstrated the polymorph changes of BCI and BCII.

60 Willey and Atalla [26] proved that the intensities of 900 cm-1 were corresponding to the disorder in the cellulose and inversely correlated to the size of the crystallites. Larger size of crystallites generates a more homogeneous molecular environment, and thus resulting narrower

-1 bands. The band at 900 cm of unmercerized BCI is significantly weaker and narrower than

that of mercerized BCII, indicating presence of higher crystallinity and larger size of crystallites in unmercerized BC. Higher intensity of band 968 cm-1 is observed in unmercerized BC, resulting from the backbone motions that are perpendicular to chain axis. Peaks at 1035 cm-1,

1095 cm-1 and 1120 cm-1 in Raman spectra of BC samples reflect the C-C and C=O stretching motions which are parallel to the chain axis [99, 106]. Significantly weaker peaks at those wave numbers are observed for mercerized BC. As it can be seen from XRD results, the crystallinity index of BC decreases from 90.1% for native BC to 60.5% of mercerized BC, further supporting that a less ordered structure was attained after mercerization.

An approach to understand the mercerization effect on BC lies in quantifying the shrinkage, which is directly correlated to microstructural changes in BC [91, 107]. Kolpak et al.

[108]reported that the threshold concentration for structure transition of plant cotton was 2.25

M and Nishiyama, Y. et al. [4] showed that shrinkage occurred in BC above 2.5 M NaOH. In this study, diametrical shrinkage in circular BC samples was observed only after exceeding

1.75 M concentration, indicating that this concentration is a transitional threshold for conversion of cellulose I to cellulose II at room temperature. Such changes in size can be

61 explained by reorganization of chains during the mercerization process to form alkali-cellulose

II with a folded-chain structure. The extent of mercerization also depends on NaOH concentration because shrinkage increased at higher NaOH concentrations. As the NaOH concentration increases hydrogen bonds are broken between cellulose chains, resulting in a more complete rearrangement of cellulose chains. From results shown in Figure 4, noticeable dimensional changes in BC fibers occurred after 5 minutes exposure to highly concentrated

NaOH demonstrating that the conversion can be achieved rapidly at room temperature. Khan et al. [85] investigated the shrinkage in dimension of jute fabrics and observed increased shrinkage with increasing reaction time and concentrations of NaOH solution, which is in agreement with those of our results of BC. However, mercerization is an irreversible reaction that cellulose II cannot be restored to the initial cellulose I after repeatedly rinsing or neutralizing with acid.

The effect of NaOH concentration on mechanical properties of different types of plant-based cellulose has been evaluated in previous studies and the mechanical enhancement varies for each fiber source. A decreasing trend in the tensile strength of mercerized plant fibers was observed in curaua [88], coir [109] and flax [110] at higher NaOH concentration. On the other hand, the tensile strength and elongation at break of the mercerized cotton yarn fiber enhanced by increasing the concentration of NaOH solution. The elastic modulus of mercerized wood fibers [111] decreased with an increase in the NaOH concentration [85]. Yet for bamboo

62 fibers, a more complex structure composed of cellulose fibers within a lignin-hemicellulose matrix, its tensile strength was maximum when mercerized with 5 M NaOH and then decreased consistently at higher NaOH content [112]. These studies focused on exploring the crystallinity to explain the differences in mechanical properties, such that different degrees of transformation from cellulose I to cellulose II is postulated to be the reason for differential response to mercerization [113].

Mechanical tests confirmed that different extents of mercerization alter the mechanical behavior of BC. In general, mercerization led to an increase in ductility, yet at the expense of tensile strength and stiffness due to the changes in crystalline structure from cellulose I to cellulose II. Native BC is composed of randomly oriented parallel aggregated glucan chains in which strong covalent bonds extend in a longitudinal direction and cross-linked by hydrogen bonds. During the mercerization, NaOH first opens fiber structure by disrupting intra-chain hydrogen bonds, and then the new generated amorphous molecular structure induces the formation of a more thermodynamically stable structure, cellulose II. After removal of NaOH by washing and drying, the antiparallel arrangement of cellulose II renders increased free volume between chains causing molecules to slide over each other more easily. Therefore, it is likely that increased molecular mobility as a result of increased NaOH concentration results in increasing extensibility.

63 Tensile strength of untreated BC is higher than those of mercerized samples. This difference is thought mainly to be due to that the swelling of cellulosic fibers increases cross section areas of microfibrils and the antiparallel chain structure further decreases the tensile force per unit area of mercerized BC can resist. Mercerized microfibrils are more likely to disentangle, reducing the capability to share load and allowing for more elastic dislocations, thereby providing a higher ductility with the loss of strength before fracture.

Results indicated that the strain energy gained by extensibility was greater than that lost by reduction in strength such that toughness at 4M or higher concentrations of NaOH was greater than unmercerized samples. It is found that mercerization can serve as an effective method to overcome the brittleness of BC. As ductility and strength are major contributions to tensile toughness values, untreated BC is stronger yet brittle with relatively lower toughness whereas mercerized BC is weaker but exhibits much higher deformability. The coiled alignment of cellulose II affects the force transmission between microfibrils which likely reduces the driving force for fracture, dissipating energy under load and providing a substantial resistance to rupture.

This study has shown that mechanical stiffness and deformability of BC can be controlled over two orders of magnitudes. Unmercerized BC had a Young’s modulus of 10 ±

1.6 GPa and 5.4 ± 1.6 % strain, close to stiff polymers and convergent to modulus of bone. In

64 contrast, Young’s modulus of mercerized BC was comparable to ductile polymers such as polyethylene or soft tissues such as tendon [114]. DIC analysis indicated that soft and hard phases can be induced continually and seamlessly by partial dipping of BC in NaOH. The DIC demonstration emulates the bone-tendon or bone ligament attachment where ductile soft tissues anchor to bone. Therefore, patterned mercerization as such may create venues for creating materials with varying mechanical stiffness or deformability over space.

BC based composites offer improved mechanical properties and have aroused interest in tissue engineering applications [115]. Instead of introducing additive to BC material, a practical technique using laser printing for fabricating mercerized patterns to design the mechanical properties of BC films has been developed. Based on our previous work, mercerization

significantly enhances the BC’s ductility by irreversibly converting BCI to BCII [101]. BCI is composed of parallel ordering of polymer chains. On the other hand, glucan chains in BCII are placed in an antiparallel structure with a lower crystallinity. Taking advantage of the

considerable difference in mechanical property between BCI and BCII, a new technique is

established to control the distribution of BCI and BCII fibers. Because dried BC films can be quite thin and fragile under the room temperature, an efficient patterning solution needs to be attachable to BC during the mercerization and separable from BC after the reaction conveniently. In this research, laser printer toner, a composite of carbon, metals and polymers, provides a good path for patterning BC. With the use of the laser printer, a thin layer of toner in pre-designed pattern is covering the BC films to shield such part from mercerization.

65 BC is considered to be a naturally isotropic material as cellulose nanofibers are oriented in all direction randomly in the culturing plane. In the natural world, bone and wood are typical anisotropic materials that the material behavior will change depending on the loading direction.

For some applications, it would be necessary to develop anisotropic material with advanced mechanical properties along a specific direction [43]. Some efforts have been devoted toward the realignment BC fibers. Tischer et al. [116] showed that using ultrasonic treatment to increase the thickness of cellulose ribbons and consequently reorganize BC fibers. Also, the control of nanofiber alignment can be achieved by directing the movement of Acetobacter xylinum cells on an artificial and oriented substrate [117]. To direct the fiber alignment, we used a laser-printing method to pattern BC samples in this work. Striped patterns of BC samples were covered by printer toner during the mercerization process, so they are composed

of the more organized structure, BCI. The rest of BC that is not covered by printer toner is exposed to the high concentration NaOH (7 M) solution and thus the microstructure is

rearranged to BCII after the treatment. BCI can be a reinforcement factor in elastic properties, as

the Young’s modulus of the BCI patterned sample is significantly higher than the modulus of

fully mercerized BC samples. The high ductility of BCII primarily contributes to the toughness by increasing the dissipation energy via cracking. With the new fiber arrangement under the effect of patterned mercerization process, the Young’s modulus and elongation at break for BC differ with change in loading direction. Since the difference of the cross-section area between

BCI and BCII part is far less than the Young’s modulus difference, elastic modulus is a

66 dominant consideration as we estimate the stiffness difference between BCI and BCII. When

BCI fibers are embedded along the axial direction, the combination of stiffness can be simplified by a model where several springs with various stiffness are connected in parallel. As

a result, BCI fibers are able to strengthen the structure of mercerized BC. This unique arrangement of microstructure leads to a relatively high Young’s modulus and less deformation

during tensile tests. When BCI fibers are oriented along the transverse direction, BCI and BCII are organized alternately in the longitudinal orientation. A model that a couple of springs are joined in series can be used to estimate the equivalent stiffness of this structure, such that a significantly smaller Young’s modulus is found in this transverse loading direction compared to that of axial loading direction. Another possible explanation is that the deformation transfers between brittle and ductile parts, as different directions of patterns offer a different path for energy dissipation. However, no significant difference is observed in UTS between the two testing directions. Therefore, the anisotropy is obtained in both Young’s modulus and elongation at break.

Figure 4.3 Comparison of FEM model and DIC result. 67

A polyester foam with re-entrant unit cells developed by Rod Lakes is the first recorded example of an artificial auxetic material [118]. The design of re-entrant honeycomb structure with a negative Poisson's ratio has been demonstrated extensively in many crystalline solids

[119].

The effect of re-entrant honeycomb patterns on BC samples has been explored with

DIC analysis. Using the laser printing technique, re-entrant honeycomb patterns consisted of an

arranged matrix of brittle BCI embed in the ductile BCII network. The geometric parameters of patterns are determined from Figure 3.1. Zhang et al. [120] investigated the relationship between Poisson’s ratio and geometric dimensions of re-entrant honeycomb patterns. Whitty et al. [121] emphasized the effect of rib thickness on the Poisson’s ratio of re-entrant honeycombs structure that the reduction of the rib thickness led to an increase in the magnitude of Poisson's ratio. The Poisson's ratio shows high dependency on the re-entrant angle and cell rib lengths. In

Eyy and Exx maps, horizontal bars move apart in the vertical direction when stretched, and the diagonal ribs exhibited positive strain expanding along the horizontal direction (Figure 4.3).

Although the lateral movement of diagonal ribs exerts tension along the x-axis on the mercerized part inside the unit cell, the interior mercerized region shrinks laterally under the axial tension load. Furthermore, horizontal bars are supposed to resist transverse contraction in

the re-entrant honeycomb structure. Even though horizontal bars consist of stiffer material BCI, they are vulnerable to the lateral compression during the test. Stress concentration is observed

68 at the junction between higher and lower modulus zones, which is consistent with the fact that the fracture always grows at the interface between mercerized zone and unmercerized zone during the tensile tests (Figure 4.4). The mechanical response of BC patterned with other re-entrant designs including arrowhead, lozenge grid, square grid and star configurations

(Figure 4.5) remain to be elucidated [122].

Figure 4.4 The rupture of the BC patterned with re-entrant honeycomb structure. Scale bar is 1 mm.

Poisson’s ratio reveals the relationship between bulk modulus B and shear modulus G:

!!!!! � = (4) !(!!!!)

For most of the materials, their Poisson's ratio range from 0 to 0.5. Material with less compressibility such as liquids and solid exhibit higher Poisson's ratio close to 0.5. Poisson's

69 ratio of compressible material such as glass and some minerals is close to 0 [123]. Poisson’s ratio is measured over a region of interest away from the clamps. The Poisson’s ratio of mercerized BC is significantly higher than that of untreated BC. Mercerized BC with unmercerized re-entrant honeycombs patterns has lower Poisson’s ratio as compared with fully mercerized BC. A possible explanation is that the Poisson’s ratio is related to the atomic packing density as a denser crystalline solid has higher Poisson’s ratio while the cellulose fibers are more compacted with a glucan chains folded structure after mercerization.

Figure 4.5 Auxetic structures achieved from different re-entrant geometries.

The patterning process is an effective way so far to control the microstructure of materials and consequently the mechanical properties of BC. Some advances in the patterning of BC have occurred by controlling the motion of living bacteria and thus to form BC films with sophisticated patterns [117, 124]. Honeycomb structured BC was fabricated by culturing

BC on a concave patterned scaffold [124]. Sano, M. B. et al. [125] verified electromagnetic

70 control of the travel pathway of bacteria during BC production. Furthermore, a photolithography process is utilized in the micropatterning of BC membrane for cell culture scaffolds [126, 127]. In our study, we patterned BC sheet by a control mercerization approach.

BCI is strong but invariably brittle, whereas mercerization provides BC with high ductility. The

BCI patterns allow for controllable distribution of the reinforcement. Patterned BC comprises hard and soft phases arranged in a customized motif to achieve control over the mechanical characteristic of BC. Additionally, pattern design is an effective approach to combine strength

and ductility. For the patterned BC sample, the BCI/BCII interface and intermolecular crosslinking enable strong bonds without complex fabrication procedures or the use of an additive crosslinking agent. Development of anisotropic BC is an example that the structural complexity for desirable properties of BC is attainable using laser printing as a feasible and fast

patterning method. Although BCI is quite stiffer than BCII, without the hollow structure, transverse expansion is not observed from the re-entrant honeycombs patterned BC when stretched in the axial direction. The possibility of controlling the Poisson’s ratio of BC with a wide variety of patterns could lead to progress in discerning the new mechanism.

A series of square patterns have been developed, exhibiting unique mechanical behaviors due to the stiffness difference between patterned parts and unpatterned parts as shown in figure 4.6. Custom patterned BC sees a wider range of applications in tissue engineering and regenerative medicine with its highly specialized microstructure and wide range of mechanical properties.

71

Figure 4.6 a, c. Geometric model. (Material assignment: Young’s modulus of the stiffer material is 700 MPa and the Young’s modulus of the softer material is 100 MPa. Boundary condition: 10% deformation in y direction.) b. Finite element results. Deformation in x direction. Shearing deformation was observed when the sample was stretched in y direction. d.

Strains in x direction. Tension was exerted in the center of the sample under stretching.

To scale up the manufacturing process for the patterning of mercerized BC, large batch production of BC can be attained by advanced bioreactors. Instead of commercial printers, high-volume printer with imposition settings is able to provide solutions for patterning in an efficient way. Although high porosity is often needed in biomaterials and scaffolds for tissue engineering (freeze-dried chitosan for example) to provide paths for fluid transport, mercerized

BC structures are more compacted with pores in the diameter of nm range. Therefore, further studies are required to address the limitation of the less porosity of mercerized BC and attain

72 mechanical stability at the same time. Another limitation of this study lies in the specimen geometry we used for mechanical tests. We did not use dumbbell shape for tensile testing specimens.

73 Appendices

A. The fabrication of BC sheet with porosity.

A cost-effective method was developed to culture surface-structured BC films. In order to generate the meshed BC, 150 polytetrafluoroethylene pillars with diameters of 1 mm were fixed on a plastic base, which has 150 blind holes drilled by CNC. Pillars were placed on the bottom of the container isometrically, and the bacteria were grown on the top of the liquid medium. The polytetrafluoroethylene mold guided the fermentation process of BC, producing a three-dimensional network of BC fibers. Further studies are needed to improve the quality of the meshed BC sheet.

Figure A. The set up used to produce BC with meshes.

74 B. The effect of temperature of mercerization on the optical properties of BC

Wavelength absorption of unmercerized BC sample and BC samples that were mercerized with 7 M NaOH at 25 °C and 90 °C with wavelength of 400 nm, 500 nm, 600 nm and 700 nm was collected. Significant difference of wavelength absorption was observed between untreated BC sample and BC sample mercerized at 90 °C.

400 nm 500 nm 600 nm 700 nm

Figure B. Wave absorption of samples mercerized at different temperatures (n =5, the wave absorption of BC samples were significantly larger than control groups, brackets connect two significantly different groups).

75 Unmercerized part Mercerized part

Figure C. A half-mercerized and half-unmercerized BC sample.

76 C. The effect of medium culture on the yield of BC

Strain Carbon Supplement Temperature Time PH Yield (g/L) Productivity Type of Reference source (°C) (day) (g/L/day) reaction Acetobacter xylinum sp. A9 Glucose none 30 7 6 2.7 0.385 Shake-flask [128] Acetobacter xylinum sp. A9 Fructose none 30 7 6 2.53 0.361 Shake-flask [128] Acetobacter xylinum sp. A9 Sucrose none 30 7 6 0.83 0.118 Shake-flask [128] Acetobacter xylinum Fructose Tomato serum broth 30 3 5 7.38±0.38 2.46±0.126 Static [129] Acetobacter xylinum Fructose+ Tomato serum broth 30 3 5 6.38±0.32 2.126±0.106 Static [129] sucrose Gluconacetobacter xylinus Glucose Hestrin Schramm medium 30 4 5 3.10 0.775 Static [130] ATCC 53524 Gluconacetobacter xylinus Sucrose Hestrin Schramm medium 30 4 5 3.83 0.975 Static [130] ATCC 53524 Gluconacetobacter xylinus Mannitol Hestrin Schramm medium 30 4 5 3.37 0.843 Static [130] ATCC 53524 Gluconacetobacter sp. A06O2 Fructose Hestrin Schramm medium 28 7 6.7 0.957 Static [131] Acetobacter xylinum NBRC Orange 2.0% peptone, 0.5% yeast 30 14 6 6.9±0.2 0.493±0.014 Static [132] 13693 juice extract and 0.12% citric acid Acetobacter xylinum NBRC Japanese 2.0% peptone, 0.5% yeast 30 14 6 4.8±0.3 0.342±0.021 Static [132] 13693 pear juice extract and 0.12% citric acid Acetobacter xylinum NBRC Grape 2.0% peptone, 0.5% yeast 30 14 6 1.4±0.2 0.1±0.014 Static [132] 13693 juice extract and 0.12% citric acid Acetobacter xylinum ATCC Konjac 5 g/L yeast extract, 3 g/L 30 8 5 Dry weight of Static [133] 23770 Powder tryptone BC 0.113±0.001g/ L Gluconacetobacter hansenii Glucose 1% (v/v) Ethanol 30 1 5 Dry weight of Shaken [134] PJK BC 2.31g/L flasks Acetobacter xylinum Fructose Corn steep liquor and 10g/L 30 3 5 BC Production Agitated [135] BPR3001A ethanol rate 0.95gŸl-1Ÿh-1 Acetobacter xylinum H2SO4-he Corn steep liquor 30 3 5 5.30 1.766 Jar [35] BPR2001 at treated fermenter molasses

77 D. Tables of data shown in figures of this work

1. The effect of striped patterns on mechanical properties of BC samples (n=5).

Young’s Modulus Ultimate Tensile Elongation at break (%)

(MPa) Strength (MPa)

Untreated BC 776 ± 135 75.9 ± 15.66 9.1 ± 1.15

Patterns in x 144 ± 20 38.5 ± 6.2 25.7 ± 8.07

direction

Patterns in y 281 ± 80 35.1 ± 14.1 13.7 ± 3.27

direction

Fully 114 ± 12 19.1 ± 3.7 16.9 ± 2.37

mercerized BC

2. Mechanical behavior of BC samples mercerized under different temperatures (n=5).

Young’s Modulus Ultimate Tensile Elongation at break (%)

(MPa) Strength (MPa)

Mercerized at 25 °C 114 ± 12 19.1 ± 3.7 16.9 ± 2.3

Mercerized at 0 °C 132 ± 21 32.1 ± 11.6 22.9 ± 6.7

Mercerized at 65 °C 147 ± 16 26.7 ± 7.8 18.7 ± 3.5

78

3. Comparison of Poisson’s ratio values (n=3).

Untreated BC Fully mercerized BC Patterned BC

Poisson’s ratio 0.32 ± 0.04 0.45 ± 0.05 0.28 ± 0.03

79 References

1. Lee, S.H., et al., The effect of bacterial cellulose membrane compared with collagen membrane on guided bone regeneration. Journal of Advanced Prosthodontics, 2015. 7(6): p. 484-495. 2. Rojas, J., Effect of Polymorphism on the Particle and Compaction Properties of Microcrystalline Cellulose. Cellulose - Medical, Pharmaceutical and Electronic Applications, 2013: p. 27-46. 3. Hussin, M.H., et al., Physicochemical of microcrystalline cellulose from oil palm fronds as potential methylene blue adsorbents. International Journal of Biological Macromolecules, 2016. 92: p. 11-19. 4. Shibazaki, H., S. Kuga, and T. Okano, Mercerization and acid hydrolysis of bacterial cellulose. Cellulose, 1997. 4(2): p. 75-87. 5. Bodin, A., et al., Influence of cultivation conditions on mechanical and morphological properties of bacterial cellulose tubes. Biotechnology and Bioengineering, 2007. 97(2): p. 425-434. 6. Czaja, W., et al., Microbial cellulose - the natural power to heal wounds. Biomaterials, 2006. 27(2): p. 145-151. 7. Malm, C.J., et al., Small calibre biosynthetic bacterial cellulose blood vessels: 13-months patency in a sheep model. Scandinavian Cardiovascular Journal, 2012. 46(1): p. 57-62. 8. Zaborowska, M., et al., Microporous bacterial cellulose as a potential scaffold for bone regeneration. Acta Biomaterialia, 2010. 6(7): p. 2540-2547. 9. Sani, A. and Y. Dahman, Improvements in the production of bacterial synthesized biocellulose nanofibres using different culture methods. Journal of Chemical Technology and Biotechnology, 2010. 85(2): p. 151-164. 10. Xu, C., et al., Bacterial Cellulose Membranes Used as Artificial Substitutes for Dural Defection in Rabbits. International Journal of Molecular Sciences, 2014. 15(6): p. 10855-10867. 11. Shoda, M. and Y. Sugano, Recent advances in bacterial cellulose production. Biotechnology and Bioprocess Engineering, 2005. 10(1): p. 1-8.

80 12. Halib, N., M.C.I.M. Amin, and I. Ahmad, Physicochemical Properties and Characterization of Nata de Coco from Local Food Industries as a Source of Cellulose. Sains Malaysiana, 2012. 41(2): p. 205-211. 13. Dufresne, C. and E. Farnworth, Tea, Kombucha, and health: a review. Food Research International, 2000. 33(6): p. 409-421. 14. Marsh, A.J., et al., Sequence-based analysis of the bacterial and fungal compositions of multiple kombucha (tea fungus) samples. Food Microbiology, 2014. 38: p. 171-178. 15. Rao, L.J.M. and K. Ramalakshmi, Recent Trends in Soft Beverages Preface. Recent Trends in Soft Beverages, 2011: p. Xiii-Xiv. 16. Aloulou, A., et al., Hypoglycemic and antilipidemic properties of kombucha tea in alloxan-induced diabetic rats. Bmc Complementary and Alternative Medicine, 2012. 12. 17. Jayabalan, R., S. Marimuthu, and K. Swaminathan, Changes in content of organic acids and tea polyphenols during kombucha tea fermentation. Food Chemistry, 2007. 102(1): p. 392-398. 18. Huang, Y., et al., Recent advances in bacterial cellulose. Cellulose, 2014. 21(1): p. 1-30. 19. Rehm, B.H.A., Bacterial polymers: biosynthesis, modifications and applications. Nature Reviews Microbiology, 2010. 8(8): p. 578-592. 20. Zogaj, X., et al., Production of cellulose and curli fimbriae by members of the family Enterobacteriaceae isolated from the human gastrointestinal tract. Infection and Immunity, 2003. 71(7): p. 4151-4158. 21. Da Re, S. and J.M. Ghigo, A CsgD-independent pathway for cellulose production and biofilm formation in Escherichia coli. Journal of Bacteriology, 2006. 188(8): p. 3073-3087. 22. Raspor, P. and D. Goranovic, Biotechnological applications of acetic acid bacteria. Critical Reviews in Biotechnology, 2008. 28(2): p. 101-124. 23. Ross, P., R. Mayer, and M. Benziman, Cellulose Biosynthesis and Function in Bacteria. Microbiological Reviews, 1991. 55(1): p. 35-58. 24. Jonas, R. and L.F. Farah, Production and application of microbial cellulose. Polymer Degradation and Stability, 1998. 59(1-3): p. 101-106. 25. Tonouchi, N., et al., Characterization of the biosynthetic pathway of cellulose from

81 glucose and fructose in Acetobacter xylinum. Bioscience Biotechnology and Biochemistry, 1996. 60(8): p. 1377-1379. 26. Hu, S.Q., et al., Structure of bacterial cellulose synthase subunit D octamer with four inner passageways. Proceedings of the National Academy of Sciences of the United States of America, 2010. 107(42): p. 17957-17961. 27. Shah, N., et al., Overview of bacterial cellulose composites: A multipurpose advanced material. Carbohydrate Polymers, 2013. 98(2): p. 1585-1598. 28. Gardner, K.H. and J. Blackwell, Structure of Native Cellulose. Biopolymers, 1974. 13(10): p. 1975-2001. 29. Vanderhart, D.L. and R.H. Atalla, Studies of Microstructure in Native Celluloses Using Solid-State C-13 Nmr. Macromolecules, 1984. 17(8): p. 1465-1472. 30. Yue, Y.Y., et al., Characterization of cellulose I/II hybrid fibers isolated from energycane bagasse during the delignification process.: Morphology, crystallinity and percentage estimation. Carbohydrate Polymers, 2015. 133: p. 438-447. 31. OSullivan, A.C., Cellulose: the structure slowly unravels. Cellulose, 1997. 4(3): p. 173-207. 32. Moon, R.J., et al., Cellulose nanomaterials review: structure, properties and nanocomposites. Chemical Society Reviews, 2011. 40(7): p. 3941-3994. 33. Klemm, D., et al., Cellulose: Fascinating biopolymer and sustainable raw material. Angewandte Chemie-International Edition, 2005. 44(22): p. 3358-3393. 34. Mamlouk, D. and M. Gullo, Acetic Acid Bacteria: Physiology and Carbon Sources Oxidation. Indian Journal of Microbiology, 2013. 53(4): p. 377-384. 35. Bae, S. and M. Shoda, Bacterial cellulose production by fed-batch fermentation in molasses medium. Biotechnology Progress, 2004. 20(5): p. 1366-1371. 36. Matsuoka, M., et al., A synthetic medium for bacterial cellulose production by Acetobacter xylinum subsp sucrofermentans. Bioscience Biotechnology and Biochemistry, 1996. 60(4): p. 575-579. 37. Yoshinaga, F., N. Tonouchi, and K. Watanabe, Research progress in production of bacterial cellulose by aeration and agitation culture and its application as a new industrial material. Bioscience Biotechnology and Biochemistry, 1997. 61(2): p. 219-224. 38. Hwang, J.W., et al., Effects of pH and dissolved oxygen on cellulose production by

82 Acetobacter xylinum BRC5 in agitated culture. Journal of Bioscience and Bioengineering, 1999. 88(2): p. 183-188. 39. Campano, C., et al., Enhancement of the fermentation process and properties of bacterial cellulose: a review. Cellulose, 2016. 23(1): p. 57-91. 40. Chawla, P.R., et al., Microbial Cellulose: Fermentative Production and Applications. Food Technology and Biotechnology, 2009. 47(2): p. 107-124. 41. Klemm, D., et al., Bacterial synthesized cellulose - artificial blood vessels for microsurgery. Progress in Polymer Science, 2001. 26(9): p. 1561-1603. 42. Amin, M.C.I.M., et al., Synthesis and characterization of thermo- and pH-responsive bacterial cellulose/acrylic acid hydrogels for drug delivery. Carbohydrate Polymers, 2012. 88(2): p. 465-473. 43. Petersen, N. and P. Gatenholm, Bacterial cellulose-based materials and medical devices: current state and perspectives. Applied Microbiology and Biotechnology, 2011. 91(5): p. 1277-1286. 44. Bodin, A., et al., Tissue-engineered conduit using urine-derived stem cells seeded bacterial cellulose polymer in urinary reconstruction and diversion. Biomaterials, 2010. 31(34): p. 8889-8901. 45. Kirdponpattara, S., et al., Structural modification and characterization of bacterial cellulose-alginate composite scaffolds for tissue engineering. Carbohydrate Polymers, 2015. 132: p. 146-155. 46. Shi, Q., et al., The osteogenesis of bacterial cellulose scaffold loaded with bone morphogenetic protein-2. Biomaterials, 2012. 33(28): p. 6644-6649. 47. Boateng, J.S., et al., Wound healing dressings and drug delivery systems: A review. Journal of Pharmaceutical Sciences, 2008. 97(8): p. 2892-2923. 48. Sulaeva, I., et al., Bacterial cellulose as a material for wound treatment: Properties and modifications. A review. Biotechnology Advances, 2015. 33(8): p. 1547-1571. 49. Solway, D.R., W.A. Clark, and D.J. Levinson, A parallel open-label trial to evaluate microbial cellulose wound dressing in the treatment of diabetic foot ulcers. International Wound Journal, 2011. 8(1): p. 69-73. 50. Kucinska-Lipka, J., I. Gubanska, and H. Janik, Bacterial cellulose in the field of wound healing and regenerative medicine of skin: recent trends and future prospectives. Polymer Bulletin, 2015. 72(9): p. 2399-2419.

83 51. Fu, L.N., J. Zhang, and G. Yang, Present status and applications of bacterial cellulose-based materials for skin tissue repair. Carbohydrate Polymers, 2013. 92(2): p. 1432-1442. 52. Helenius, G., et al., In vivo biocompatibility of bacterial cellulose. Journal of Biomedical Materials Research Part A, 2006. 76a(2): p. 431-438. 53. Pertile, R.A.N., et al., Surface modification of bacterial cellulose by nitrogen-containing plasma for improved interaction with cells. Carbohydrate Polymers, 2010. 82(3): p. 692-698. 54. Ravi, S. and E.L. Chaikof, Biomaterials for vascular tissue engineering. Regenerative Medicine, 2010. 5(1): p. 107-120. 55. Fink, H., et al., Real-time measurements of coagulation on bacterial cellulose and conventional vascular graft materials. Acta Biomaterialia, 2010. 6(3): p. 1125-1130. 56. Backdahl, H., et al., Mechanical properties of bacterial cellulose and interactions with smooth muscle cells. Biomaterials, 2006. 27(9): p. 2141-2149. 57. Schumann, D.A., et al., Artificial vascular implants from bacterial cellulose: preliminary results of small arterial substitutes. Cellulose, 2009. 16(5): p. 877-885. 58. Scherner, M., et al., In vivo application of tissue-engineered blood vessels of bacterial cellulose as small arterial substitutes: proof of concept? Journal of Surgical Research, 2014. 189(2): p. 340-347. 59. Klechkovskaya, V.V., et al., Network model of Acetobacter xylinum cellulose intercalated by drug nanoparticles. Nanomaterials for Applications in Medicine and Biology, 2008: p. 165-177. 60. Almeida, I.F., et al., Bacterial cellulose membranes as drug delivery systems: An in vivo skin compatibility study. European Journal of Pharmaceutics and Biopharmaceutics, 2014. 86(3): p. 332-336. 61. Bodhibukkana, C., et al., Composite membrane of bacterially-derived cellulose and molecularly imprinted polymer for use as a transdermal enantioselective controlled-release system of racemic propranolol. Journal of Controlled Release, 2006. 113(1): p. 43-56. 62. Silva, N.H.C.S., et al., Topical caffeine delivery using biocellulose membranes: a potential innovative system for cellulite treatment. Cellulose, 2014. 21(1): p. 665-674. 63. Trovatti, E., et al., Bacterial cellulose membranes applied in topical and transdermal

84 delivery of lidocaine hydrochloride and ibuprofen: In vitro diffusion studies. International Journal of Pharmaceutics, 2012. 435(1): p. 83-87. 64. Chan, B.P. and K.W. Leong, Scaffolding in tissue engineering: general approaches and tissue-specific considerations. European Spine Journal, 2008. 17: p. S467-S479. 65. Swetha, M., et al., Biocomposites containing natural polymers and hydroxyapatite for bone tissue engineering. International Journal of Biological Macromolecules, 2010. 47(1): p. 1-4. 66. Burg, K.J.L., S. Porter, and J.F. Kellam, Biomaterial developments for bone tissue engineering. Biomaterials, 2000. 21(23): p. 2347-2359. 67. Mendes, P.N., et al., In vivo and in vitro evaluation of an Acetobacter xylinum synthesized microbial cellulose membrane intended for guided tissue repair. Acta Veterinaria Scandinavica, 2009. 51. 68. Brackmann, C., et al., In situ Imaging of Collagen Synthesis by Osteoprogenitor Cells in Microporous Bacterial Cellulose Scaffolds. Tissue Engineering Part C-Methods, 2012. 18(3): p. 227-234. 69. Mori, R., et al., Increased Antibiotic Release from a Bone Cement Containing Bacterial Cellulose. Clinical Orthopaedics and Related Research, 2011. 469(2): p. 600-606. 70. Hu, Y., et al., Engineering of porous bacterial cellulose toward human fibroblasts ingrowth for tissue engineering. Journal of Materials Research, 2014. 29(22): p. 2682-2693. 71. Gao, C.A., et al., Preparation and characterization of bacterial cellulose sponge with hierarchical pore structure as tissue engineering scaffold. Journal of Porous Materials, 2011. 18(2): p. 139-145. 72. Baah-Dwomoh, A., et al., The feasibility of using irreversible electroporation to introduce pores in bacterial cellulose scaffolds for tissue engineering. Applied Microbiology and Biotechnology, 2015. 99(11): p. 4785-4794. 73. O'Driscoll, S.W., The healing and regeneration of articular cartilage. Journal of Bone and Joint Surgery-American Volume, 1998. 80a(12): p. 1795-1812. 74. Moutos, F.T. and F. Guilak, Composite scaffolds for cartilage tissue engineering. Biorheology, 2008. 45(3-4): p. 501-512. 75. Cao, Z., C. Dou, and S.W. Dong, Scaffolding Biomaterials for Cartilage Regeneration.

85 Journal of Nanomaterials, 2014. 76. Martinez, H., et al., Mechanical stimulation of fibroblasts in micro-channeled bacterial cellulose scaffolds enhances production of oriented collagen fibers. Journal of Biomedical Materials Research Part A, 2012. 100a(4): p. 948-957. 77. Ahrem, H., et al., Laser-structured bacterial nanocellulose hydrogels support ingrowth and differentiation of chondrocytes and show potential as cartilage implants. Acta Biomaterialia, 2014. 10(3): p. 1341-1353. 78. Feldmann, E.M., et al., Description of a novel approach to engineer cartilage with porous bacterial nanocellulose for reconstruction of a human auricle. Journal of Biomaterials Applications, 2013. 28(4): p. 626-640. 79. Avila, H.M., et al., Biocompatibility evaluation of densified bacterial nanocellulose hydrogel as an implant material for auricular cartilage regeneration. Applied Microbiology and Biotechnology, 2014. 98(17): p. 7423-7435. 80. Subramanian, A., U.M. Krishnan, and S. Sethuraman, Development of biomaterial scaffold for nerve tissue engineering: Biomaterial mediated neural regeneration. Journal of Biomedical Science, 2009. 16. 81. Kowalska-Ludwicka, K., et al., Modified bacterial cellulose tubes for regeneration of damaged peripheral nerves. Archives of Medical Science, 2013. 9(3): p. 527-534. 82. KroonBatenburg, L.M.J. and J. Kroon, The crystal and molecular structures of cellulose I and II. Glycoconjugate Journal, 1997. 14(5): p. 677-690. 83. Kaith, B.S., et al., Mercerization of Flax Fiber Improves the Mechanical Properties of Fiber-Reinforced Composites. International Journal of Polymeric Materials, 2008. 57(1): p. 54-72. 84. Das, M., A. Pal, and D. Chakraborty, Effects of mercerization of bamboo strips on mechanical properties of unidirectional bamboo - Novolac composites. Journal of Applied Polymer Science, 2006. 100(1): p. 238-244. 85. Khan, J.A., M.A. Khan, and R. Islam, Effect of Mercerization on Mechanical, Thermal and Degradation Characteristics of Jute Fabric-reinforced Polypropylene Composites. Fibers and Polymers, 2012. 13(10): p. 1300-1309. 86. Wang, W.M., et al., Changes in Composition, Structure, and Properties of Jute Fibers after Chemical Treatments. Fibers and Polymers, 2009. 10(6): p. 776-780. 87. Yue, Y.Y., G.P. Han, and Q.L. Wu, Transitional Properties of Cotton Fibers from

86 Cellulose I to Cellulose II Structure. Bioresources, 2013. 8(4): p. 6460-6471. 88. Gomes, A., K. Goda, and J. Ohgi, Effects of alkali treatment to reinforcement on tensile properties of curaua fiber green composites. Jsme International Journal Series a-Solid Mechanics and Material Engineering, 2004. 47(4): p. 541-546. 89. Nakagaito, A.N. and H. Yano, Toughness enhancement of cellulose nanocomposites by alkali treatment of the reinforcing cellulose nanofibers. Cellulose, 2008. 15(2): p. 323-331. 90. Siro, I. and D. Plackett, Microfibrillated cellulose and new nanocomposite materials: a review. Cellulose, 2010. 17(3): p. 459-494. 91. Revol, J.F., A. Dietrich, and D.A.I. Goring, Effect of Mercerization on the Crystallite Size and Crystallinity Index in Cellulose from Different Sources. Canadian Journal of Chemistry-Revue Canadienne De Chimie, 1987. 65(8): p. 1724-1725. 92. Saska, S., et al., Bacterial cellulose-collagen nanocomposite for bone tissue engineering. Journal of Materials Chemistry, 2012. 22(41): p. 22102-22112. 93. Cai, Z.J. and G. Yang, Bacterial Cellulose/Collagen Composite: Characterization and First Evaluation of Cytocompatibility. Journal of Applied Polymer Science, 2011. 120(5): p. 2938-2944. 94. Wang, J., et al., Immobilization of gelatin on bacterial cellulose nanofibers surface via crosslinking technique. Materials Science & Engineering C-Materials for Biological Applications, 2012. 32(3): p. 536-541. 95. Lin, W.C., et al., Bacterial cellulose and bacterial cellulose-chitosan membranes for wound dressing applications. Carbohydrate Polymers, 2013. 94(1): p. 603-611. 96. Ostadhossein, F., et al., Development of Chitosan/Bacterial Cellulose Composite Films Containing Nanodiamonds as a Potential Flexible Platform for Wound Dressing. Materials, 2015. 8(9): p. 6401-6418. 97. Kingkaew, J., et al., Effect of molecular weight of chitosan on antimicrobial properties and tissue compatibility of chitosan-impregnated bacterial cellulose films. Biotechnology and Bioprocess Engineering, 2014. 19(3): p. 534-544. 98. Li, Y., et al., Bacterial cellulose-hyaluronan nanocomposite biomaterials as wound dressings for severe skin injury repair. Journal of Materials Chemistry B, 2015. 3(17): p. 3498-3507. 99. Wiley, J.H. and R.H. Atalla, Band Assignments in the Raman-Spectra of Celluloses.

87 Carbohydrate Research, 1987. 160: p. 113-129. 100. Okano, T. and A. Sarko, Mercerization of Cellulose .2. Alkali Cellulose Intermediates and a Possible Mercerization Mechanism. Journal of Applied Polymer Science, 1985. 30(1): p. 325-332. 101. Kolpak, F.J. and J. Blackwell, Mercerization of Cellulose .2. Morphology of Mercerized Cotton Cellulose. Polymer, 1978. 19(2): p. 132-135. 102. Nishimura, H. and A. Sarko, Mercerization of Cellulose .3. Changes in Crystallite Sizes. Journal of Applied Polymer Science, 1987. 33(3): p. 855-866. 103. Takai, M. and J.R. Colvin, Mechanism of Transition between Cellulose-I and Cellulose-Ii during Mercerization. Journal of Polymer Science Part a-Polymer Chemistry, 1978. 16(6): p. 1335-1342. 104. Nishiyama, Y., S. Kuga, and T. Okano, Mechanism of mercerization revealed by X-ray diffraction. Journal of Wood Science, 2000. 46(6): p. 452-457. 105. Dinand, E., et al., Mercerization of primary wall cellulose and its implication for the conversion of cellulose I -> cellulose II. Cellulose, 2002. 9(1): p. 7-18. 106. Schenzel, K. and S. Fischer, NIR FT Raman spectroscopy - a rapid analytical tool for detecting the transformation of cellulose polymorphs. Cellulose, 2001. 8(1): p. 49-57. 107. Langan, P., Y. Nishiyama, and H. Chanzy, X-ray structure of mercerized cellulose II at 1 angstrom resolution. Biomacromolecules, 2001. 2(2): p. 410-416. 108. Kolpak, F.J., M. Weih, and J. Blackwell, Mercerization of Cellulose .1. Determination of Structure of Mercerized Cotton. Polymer, 1978. 19(2): p. 123-131. 109. Gu, H., Tensile behaviours of the coir fibre and related composites after NaOH treatment. Materials & Design, 2009. 30(9): p. 3931-3934. 110. Lazic, B.D., et al., Effect of chemical treatments on the chemical composition and properties of flax fibers. Journal of the Serbian Chemical Society, 2017. 82(1): p. 83-97. 111. Tanimoto, T. and T. Nakano, Difference in reduction properties between longitudinal dimension and elastic modulus of wood induced with aqueous NaOH treatment: modeling and analysis. Journal of Wood Science, 2016. 62(1): p. 12-19. 112. Das, M. and D. Chakraborty, Evaluation of improvement of physical and mechanical properties of bamboo fibers due to alkali treatment. Journal of Applied Polymer Science, 2008. 107(1): p. 522-527.

88 113. Liu, Y.P. and H. Hu, X-ray diffraction study of bamboo fibers treated with NaOH. Fibers and Polymers, 2008. 9(6): p. 735-739. 114. Qiu, K.Y. and A.N. Netravali, A Review of Fabrication and Applications of Bacterial Cellulose Based Nanocomposites. Polymer Reviews, 2014. 54(4): p. 598-626. 115. Abeer, M.M., M.C.I.M. Amin, and C. Martin, A review of bacterial cellulose-based drug delivery systems: their biochemistry, current approaches and future prospects. Journal of Pharmacy and Pharmacology, 2014. 66(8): p. 1047-1061. 116. Tischer, P.C.S.F., et al., Nanostructural Reorganization of Bacterial Cellulose by Ultrasonic Treatment. Biomacromolecules, 2010. 11(5): p. 1217-1224. 117. Kondo, T., et al., Biodirected epitaxial nanodeposition of polymers on oriented macromolecular templates. Proceedings of the National Academy of Sciences of the United States of America, 2002. 99(22): p. 14008-14013. 118. Lakes, R., Foam Structures with a Negative Poissons Ratio. Science, 1987. 235(4792): p. 1038-1040. 119. Masters, I.G. and K.E. Evans, Models for the elastic deformation of honeycombs. Composite Structures, 1996. 35(4): p. 403-422. 120. Zhang, X.W. and D.Q. Yang, Mechanical Properties of Auxetic Cellular Material Consisting of Re-Entrant Hexagonal Honeycombs. Materials, 2016. 9(11). 121. Whitty, J.P.M., F. Nazare, and A. Alderson, Modelling the effects of density variations on the in-plane Poisson's ratios and Young's moduli of periodic conventional and re-entrant honeycombs - Part 1: Rib thickness variations. Cellular Polymers, 2002. 21(2): p. 69-98. 122. Saxena, K.K., R. Das, and E.P. Calius, Three Decades of Auxetics Research - Materials with Negative Poisson's Ratio: A Review. Advanced Engineering Materials, 2016. 18(11): p. 1847-1870. 123. Pohle, F.V., Foundations of Solid Mechanics. Journal of the Franklin Institute-Engineering and Applied Mathematics, 1966. 281(4): p. 360-&. 124. Uraki, Y., et al., Honeycomb-like architecture produced by living bacteria, Gluconacetobacter xylinus. Carbohydrate Polymers, 2007. 69(1): p. 1-6. 125. Sano, M.B., et al., Electromagnetically Controlled Biological Assembly of Aligned Bacterial Cellulose Nanofibers. Annals of Biomedical Engineering, 2010. 38(8): p. 2475-2484.

89 126. Karita, Y., et al., Micropatterning of Bacterial Cellulose as Degradable Substrate for Cell Culture. 2014 Ieee 27th International Conference on Micro Electro Mechanical Systems (Mems), 2014: p. 518-519. 127. Geisel, N., et al., Microstructured Multilevel Bacterial Cellulose Allows the Guided Growth of Neural Stem Cells. Small, 2016. 12(39): p. 5407-5413. 128. Son, H.J., et al., Optimization of fermentation conditions for the production of bacterial cellulose by a newly isolated Acetobacter sp A9 in shaking cultures. Biotechnology and Applied Biochemistry, 2001. 33: p. 1-5. 129. Embuscado, M.E., J.S. Marks, and J.N. Bemiller, Bacterial Cellulose .1. Factors Affecting the Production of Cellulose by Acetobacter-Xylinum. Food Hydrocolloids, 1994. 8(5): p. 407-418. 130. Mikkelsen, D., et al., Influence of different carbon sources on bacterial cellulose production by Gluconacetobacter xylinus strain ATCC 53524. Journal of Applied Microbiology, 2009. 107(2): p. 576-583. 131. Karahan, A.G., et al., Some Properties of Bacterial Cellulose Produced by New Native Strain Gluconacetobacter sp. A06O2 Obtained from Turkish Vinegar. Journal of Applied Polymer Science, 2011. 121(3): p. 1823-1831. 132. Kurosumi, A., et al., Utilization of various fruit juices as carbon source for production of bacterial cellulose by Acetobacter xylinum NBRC 13693. Carbohydrate Polymers, 2009. 76(2): p. 333-335. 133. Hong, F. and K.Y. Qiu, An alternative carbon source from konjac powder for enhancing production of bacterial cellulose in static cultures by a model strain Acetobacter aceti subsp xylinus ATCC 23770. Carbohydrate Polymers, 2008. 72(3): p. 545-549. 134. Park, J.K., J.Y. Jung, and Y.H. Park, Cellulose production by Gluconacetobacter hansenii in a medium containing ethanol. Biotechnology Letters, 2003. 25(24): p. 2055-2059. 135. Naritomi, T., et al., Effect of ethanol on bacterial cellulose production from fructose in continuous culture. Journal of Fermentation and Bioengineering, 1998. 85(6): p. 598-603.

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