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).

(!!!" !!!" )
                       𝐶𝑟𝐼 = !!!"
×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 (%) (MJŸm-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
   3.8 Typical XRD spectra for untreated BC (BCI) and mercerized BC (BCII) films.
Figure
 

 
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 (%)
c. d.

Stress (MPa)

 
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

bands. The band at 900 cm-1 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:

!!!!!
𝜈 = !(!!!!) (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
0.95gŸl-1Ÿh-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  
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