Master Thesis Bacterial Cellulose and Thermoplastics

Master Thesis Bacterial Cellulose and Thermoplastics

(Parte 1 de 4)

By ELVIE ESCORRO BROWN

A thesis submitted in partial fulfillment of the requirements for the degree of

WASHINGTON STATE UNIVERSITY Department of Chemical Engineering

MAY 2007

To the Faculty of Washington State University:

The members of the Committee appointed to examine the thesis of

ELVIE ESCORRO BROWN find it satisfactory and recommend that it be accepted.

_ Chair

Firstly, I would like to thank my advisor, Marie-Pierre Laborie for giving me the chance to work on a very interesting project. She has given me the freedom to explore on my own but her brilliant ideas and guidance never left me adrift. I am very grateful to my committee, James Lee and Eric Aston who shared their knowledge and imparted significant assistance in finishing my research. A special thanks goes to Bernard Van Wie for encouraging me to try M.S. degree, I never thought I’d go this far.

All of the WMEL personnel have become important to me not only for the help in my project but also for the friendship. For the two years that I was here, I bonded with impeccably wonderful people. Thank you very much and I hope to keep the communications with you.

I thank International Marketing Program for Agricultural Commodities and Trade

(IMPACT) for providing financial support for my research and also for featuring me in their newsletter. I also would like to thank Society of Women Engineers (SWE) for the support during my first year of MS degree.

I do believe that there should be a bigger word than ‘thank you’ for the man who’s always been there for all my highs and lows and the reason why I’m in this country; my husband, Alan. His unfading support has given me the strength and enthusiasm (my own drive amazes me sometimes).

I would like to express my heartfelt gratitude to my Mom and Dad, Loretta and

Earl Brown; my Mama who’s in the Philippines, Benita Escorro and the rest of the family for the encouragement and pride. They made me believe in myself. Success is sweet because of all of these wonderful people.

Most importantly, I like to thank God for everything, for I believe that his sacred blessings put me where I am right now.

Abstract by Elvie E. Brown, M.S.

Washington State University May 2007

Chair: Marie Pierre Laborie

Bacterial cellulose (BC) has many applications in membranes, electronics, textiles and especially in the biomedical field. For bacterial cellulose to be more effectively utilized in these applications, it is imperative to fine-tune its properties.

This research aims at engineering the morphology, composition and structure of

BC-thermoplastic polymer nanocomposites by augmenting the growth medium of the cellulose-producing bacterium, Acetobacter xylinum, with the thermoplastic polymer, thereby manipulating BC biogenesis. It is hypothesized that addition of the thermoplastic polymer into the medium allows the development of intermolecular interactions with the BC fibers during cellulose crystallization into nanofibers, yielding a thermoplastic nanocomposite reinforced with finely dispersed BC nanofibers.

Engineering of the nanocomposites was done by varying the type or the amount of polymer added to the medium. Varying polymers by addition of poly(ethylene oxide) (PEO) or poly(vinyl alcohol) (PVA) into the medium both produced nanocomposites with dispersed BC nanofibers and showed polymer melting point depression. However, BC/PVA nanocomposite demonstrates considerable miscibility and interaction when compared to BC-PEO material by forming hydrogen bonds and also by having single glass transition and degradation temperatures. Varying the amount of polymer added into the medium varied all the characterized properties. BC fiber dispersity, polymer chemical composition and nanocomposite surface roughness increased as polymer amount in medium increased. Also, thermal and mechanical stability increased as BC loading in the nanocomposite increased. These variations of properties illustrated that BC/PEO and BC/PVA nanocomposites can be engineered by BC biogenesis manipulation.

Page

CHAPTER I: INTRODUCTION1
Applications of Bacterial Cellulose1
Biogenesis of BC4
Modification of BC6
Research Objectives9
References10
CHAPTER I: BIOENGINEERING OF BC/PEO NANOCOMPOSITES15
Introduction15
Materials and Methods18
Production of the Starter Culture18
Transmission Electron Microscopy (TEM)19
Atomic Force Microscopy (AFM)20
Thermogravimetric Analysis (TGA)20
Fourier Transform Infrared Spectroscopy (FTIR)20

ACKNOWLEDGMENT…………………………………………………………………...i ABSTRACT………………………………………………………………………………..iv LIST OF TABLES………………………………………………………………………….x LIST OF FIGURES……………………………………………………………………….xii Production of Bacterial Cellulose into Polyethylene Oxide Modified Media18 Differential Scanning Calorimetry (DSC)......................................................21

Dynamic Mechanical Analysis (DMA)2
Results and Discussion2
Morphology of Cellulose/PEO Nanocomposites2
Tailoring the Chemical Composition of Cellulose/PEO Nanocomposites26
Physical and Mechanical Properties of BC/PEO Nanocomposites36
Conclusions38
References39
CHAPTER I: BIOENGINEERING OF BC/PVA NANOCOMPOSITES4
Introduction4
Materials and Methods46
Production of the Starter Culture46
Production of BC in the Poly(vinyl alcohol)-Modified HS Media47
Transmission Electron Microscopy (TEM)48
Atomic Force Microscopy (AFM)49
Fourier-Transform Infrared Spectroscopy (FT-IR)49
Thermogravimetric Analysis (TGA)50
Dynamic Scanning Calorimetry (DSC)51
Dynamic Mechanical Analysis (DMA)51
Results and Discussion52
Production Results52
Morphology of BC/PVA Nanocomposites54

vii Chemical Compositions of BC/PVA Nanocomposites..................................58 viii

Nanocomposites and Its Influence to Thermal Properties60
Mechanical Properties of BC/PVA Nanocomposites71
Conclusion73
References75
CHAPTER IV: CONCLUSION78
Summary of Research Findings78
Future Works81
References83
NANOCOMPOSITES85
Images of Production setup85
Images of Dried Products8
APPENDIX B: BC/PEO NANOCOMPOSITE DATA89
TEM Images90
AFM Images92
Computation of Equilibrium Melting Temperature95
Calculation of χ1296
Production Yield98
APPENDIX C: BC/PVA NANOCOMPOSITE DATA9
TEM Images100

Evaluation of Molecular Interaction and Crystallinity of BC/PVA APPENDIX A: PRODUCTION OF BC/THERMOPLASTIC POLYMER AFM Images.............................................................................................................102

FT-IR Data104
Production Yield105

ix Solubility Parameter Computations..........................................................................105

Table I-1. Bacterial cellulose applications3
Table I-2. Modification of BC Biogenesis9
and polyethylene oxide (PEO) and their nanocomposites27

Page Table I-1. Degradation temperature and chemical composition of bacterial cellulose (BC)

(PEO) and bacterial cellulose (BC) in nanocomposites of varying BC:PEO ratios32
Table I-1. Solubility parameters46
Table I-2. Density of BC, PVA and BC/PVA samples53
Table I-3. Composition of BC/PVA nanocomposites60
Table I-4. FT-IR characteristic peaks of BC, PVA and BC/PVA samples61
materials65
Table I-6.Tm and Tg data from DSC67
Table I-7.Storage modulus, Tg and Tm data of BC/PVA nanocomposites from DMA72
Table IV-1. BC-PEO nanocomposite properties79
Table IV-2. BC-PVA nanocomposite properties79
Table IV-3. Ways of improving BC production83
Table B-1. Wt% conversion of D-glucose to BC and PEO1 to nanocomposite98

Table I-2. Thermal transitions and other morphological characteristics of polyethylene oxide Table I-5. Degradation temperatures obtained from TGA for BC, PVA and BC/PVA Table C-1. Known cellulose wt% and A1165/A850 values for calibration to use for compositional analysis of produced BC/PVA nanocomposites.................................104

xii

Figure I-1. Mechanism of BC formation by Acetobacter xylinum4

Page Figure I-1. TEM images (60K) of bacterial cellulose/polyethylene oxide (BC/PEO) products:

medium with 3% PEO and d) HS medium with 5% PEO23

BC grown in a) Hestrin-Shramm (HS) medium, b) HS medium with 1% PEO, c) HS

HS medium with 5% PEO25

Figure I-2. Atomic force microscopy(AFM) topographical images (3x3 µm) of bacterial cellulose/ polyethylene oxide (BC/PEO) products obtained in a) Hestrin-Shramm (HS) medium, b) HS medium with 1% PEO, c) HS medium with 3% PEO and d)

nanocomposites as a function of the culture medium modification (bottom)27

Figure I-3. Thermogravimetric analysis illustrating the original and derivative curves for the pure cellulose (BC) and polyethylene oxide (PEO) (top) and for all the BC/PEO

nanocomposites as a function of BC:PEO w/w ratio30

Figure I-4. FTIR spectra of bacterial cellulose (BC)/ polyethylene oxide (PEO)

BC:PEO w/w ratio (right)31

Figure I-5. Differential scanning calorimetry thermograms illustrating the determination of the glass transition and melting temperatures in the nanocomposite (left) and the variation in the melting endotherm of polyethylene oxide (PEO) as a function of the

Figure I-6. Hoffman-Weeks plots for determining the equilibrium melting temperatures in control polyethylene oxide (PEO) and in a bacterial cellulose (BC)/PEO nanocomposite. ............................................................................................................3 xiii

nanocomposites as a function of BC:PEO w/w ratio37

Figure I-7. Root mean square roughness bacterial cellulose (BC)/polyethylene oxide (PEO)

varying BC:PEO w/w ratios38
Figure I-1. Molecular Structures of cellulose and PVA45
Figure I-2. Actual and calculated weighted average density vs. PVA initial amount53
medium with B)1wt% PVA C) 5wt% PVA D) 9wt% PVA5

Figure I-8. Storage tensile modulus E’ versus temperature at 1 Hz for nanocomposites of Figure I-3. TEM Images of BC and PVA modified BC. A)control BC. BC grown in

B)1wt%PVA, ribbon width=61±6nm C)5wt% PVA D)9wt% PVA57
Figure I-5. Calibration and product data from FT-IR for chemical composition analysis59
highlighted with dotted lines62
Figure I-7. TGA data of BC, PVA and BC/PVA nanocomposites64
Figure I-8. Derivative data of TGA. Tdeg of PVA is pointed out by the dotted line65
highlighted with dotted lines and glass transition temperatures (Tg) with arrows6

Figure I-4. AFM image. A)BC, ribbon width=104±16nm. BC grown in HS medium with Figure I-6. FT-IR spectra of BC, PVA and BC/PVA samples. Characteristic peaks are Figure I-9. DSC data of PVA, BC and BC/PVA samples. Melting temperature (Tm) is Figure I-10. Gordon-Taylor Equation fitting to BC-PVA products and Nishio and Manley

(1988) wood pulp cellulose-PVA data70

Figure I-1. DMA data of BC/PVA nanocomposites. Arrows pointed out Tm of samples...72 xiv

regeneration (Seal et al 2001)82
Figure A-1. Incubation in magnetically-stirred environment86
Figure A-2. Stringy material adhered to the Teflon stirrer instigate growth of product86
the product87
Figure A-4. Product ready for harvest87
Figure A-5. Freeze-dried and flattened nanocomposites8
Figure B-1. TEM images of BC in unmodified HS medium90
Figure B-2. TEM images of BC in 1wt% PEO1-modified HS medium90
Figure B-3. TEM images of BC in 3wt% PEO1-modified HS medium91
Figure B-4. TEM images of BC in 5wt% PEO1-modified HS medium91
Figure B-5. AFM image of dried BC grown in unmodified HS medium92
Figure B-6. AFM image of dried BC grown in unmodified HS medium93
Figure B-7. AFM Images of dried BC grown in 1wt% PEO1-modified HS medium93
Figure B-8. AFM Images of dried BC grown in 3wt% PEO1-modified HS medium94
Figure B-9. AFM Images of dried BC grown in 5wt% PEO1-modified HS medium94
Figure C-1. TEM Images of BC grown in unmodified HS medium100
Figure C-2. TEM Images of BC grown in 1wt% PVA-modified HS medium100
Figure C-3. TEM Images of BC grown in 5wt% PVA-modified HS medium101

Figure IV-1. Illustration of how some material, biological, medical and engineering properties must be integrated to achieve successful biomaterials for tissue Figure A-3. Image of the bottom of the Erlenmeyer flask. The white cotton-like material is Figure C-4. TEM Images of BC grown in 9wt% PVA-modified HS medium......................101

Figure C-5. AFM images of dried BC grown in 1wt% PVA-modified HS medium102
Figure C-6. AFM Images of dried BC grown in 5wt% PVA-modified HS medium102
Figure C-7. AFM Images of dried BC grown in 9wt% PVA-modified HS medium103

Figure C-8. A1165/A850 from FT-IR data of microcrystalline cellulose/PVA blend as calibration to use for composition analysis of the produced BC/PVA nanocomposites.

xvi

Dedication For Alan

Applications of Bacterial Cellulose

Bacterial Cellulose (BC) has gained attention in the research realm for the favorable properties it possesses; such as its remarkable mechanical properties in both dry and wet states, porosity, water absorbency, moldability, biodegrability and excellent biological affinity (Shoda and Sugano 2005). Because of these properties, BC has a wide range of potential applications including use as a separation medium for water treatment (Brown 1989, Choi et al 2004), a specialty carrier for battery fluids and fuel cells (Brown 1989), a mixing agent, a viscosity modifier (Brown 1989, Jonas and Farah 1998), light transmitting optical fibers (Brown 1989), a biological substrate medium (Brown 1989, Watanabe et al 1993), food or food substitute (Miranda et al 1965, Brown 1989, Jonas and Farah 1998), lint-free specialty clothing (Brown 1989), optoelectronics devices (Nogi et al 2005), paper (Jonas and Farah 1998, Shah and Brown 2005), stereo diaphragms (Jonas and Farah 1998) and immobilization matrices of proteins or chromatography substances (Jonas and Farah 1998, Sokolnicki et al 2006). The prevalent application of BC is in the biomedical field, as it is highly useful for wound dressing (Hamlyn et al 1997, Cienchanska 2004, Legeza et al 2004, Wan and Millon 2005, Czaja et al 2006); artificial skin (Jonas and Farah 1998, Czaja et al 2007); dental implants; vascular grafts; catheter covering dressing (Wan and Millon 2005); dialysis membrane (Wan and Millon 2005, Sokolnicki et al 2006); coatings for cardiovascular stents, cranial stents (Wan and Millon 2005), membranes for tissue-guided regeneration (Wan and Millon 2005, Czaja et al

2007), tissue replacement, controlled-drug release carriers (Wan and Millon 2005), vascular prosthetic devices (Charpentier et al 2006), a scaffold for tissue engineering (Czaja et al 2007), and as artificial blood vessels (Klemm et al 2001, Backdahl et al 2006, Wan et al 2006). For BC to be suitable for these diverse applications, some of its properties must be modified. Modification of BC had been accomplished in the applications listed in Table I-1.

Applications BC Product Processing Ideal/Obtained Properties References

Vascular prosthetic device (to replace diseased arteries)

-BC films are used to coat surface-treated medicalgrade polyesters.

-Minimizes blood clotting and increases biocompatibility. -Has high mechanical strength in wet state, substantial permeability to water and gases, high water retention and low surface roughness.

(Charpentier et al, 2006)

Wound care product (wounds such as thermal burns)

-BC sheets were impregnated with drugs known as SOD (procel- Super), porviargol(Procel- PA) and Inerpan. -Never-dried BC sheets are immersed in chitosan solution. -BC grown statically in a chitosan-modified medium.

-Highly nanoporous, allowing transfer of antibiotics or medicines while serving as a physical barrier against external infections. -Wound healing accelerated.

-High mechanical properties in wet state

(Hamlyn et al 1997,

Ciechanska 2004,

Legeza et al 2004, Czaja et al 2006)

Artificial blood vessel in microsurgery

-BC grown in a static culture molded in BASYC® tubes (a hollow-shaped tube mimicking blood vessel shape).

-High mechanical strength in wet state enormous water retention values, low surface roughness of inner surface. -Highly moldable in situ.

-Can sustain a mean tensile force of 800mN.

(Klemm et al, 2001)

Tissue-engineered blood vessels

-BC grown in a tubularshaped mold.

-Young modulus should match carotid arteries, about 3MPa. -Inner side of tubular BC must be smoother compared to the outside.

(Backdahl et al, 2006)

Optically transparent reinforcement for optoelectronics industry (for transparent polymers used for displays)

-BC sheets are impregnated with acrylic resins.

-Highly transparent due to its nanoscale fibers free from light scattering. -Low thermal coefficient.

-Mechanical strength 5 times that of engineered plastics.

Substrate for mammalian cell culture

-BC membrane grown statically and electrically charged.

-High permeability (Watanabe et al, 1993)

Applications BC Product Processing Ideal/Obtained Properties References

Cation-exchange membrane for industrial wastewater treatment

-BC membranes are modified with cationexchangeable acrylic acid

-Tensile strength of 12MPa and elongation of 6% (Choi et al, 2004)

Electronic paper -BC sheets are doped with conductors for embedding of electronic dyes.

-Paper has high reflectivity and contrast. -Improved conductivity in BC.

Encapsulation membrane system for living tissues or protein enzymes

-Statically grown BC membrane.

-BC have the appropriate mass transfer parameters and membrane morphology

(Sokolnicki et al, 2006)

Table I-1. Bacterial cellulose applications.

Table I-1 illustrates that for specific applications, specific properties must be met.

Note that from the applications mentioned above, and in Table I-1, biomedical dominion has substantial utilization. The need for biomedical materials has grown significantly over the years (Anderson 2006, Jagur-Grodzinski 2006) and for this need, BC is highly regarded for it has the suitable properties especially for regenerative medicine (Czaja et al 2007). Yet for biomedical application, properties such as thermal stability, strength, porosity, roughness, morphology and density are crucial (Rezwan et al 2006). Fine-tuning of these properties is imperative for BC to conform to the substituted environment (Jagur- Grodzinski 2006, Rezwan et al 2006). Table I-1 cites some of the necessitated modifications for BC properties. In this research, modification is directed at the biogenesis of BC.

In the next section, the synthesis of BC by bacterium Acetobacter xylinum is discussed, to provide insight into the plausible property fine-tuning method of BC.

Biogenesis of BC

Figure I-1. Mechanism of BC formation by Acetobacter xylinum.

BC is a product of microbes’ primary metabolic processes. It is produced by a species of Zoogloea, Sarcina, (Canale-Parola and Wolfe 1960), Salmonella, Rhizobium (Napoli et al 1975), Pseudomonas (Spiers et al 2003), Escherichia, Agrobacterium, (Matthysse et al 1995), Aerobacter, Achromobacter, Azotobacter, Alcaligenes, and Acetobacter. The most studied and most used BC-producing bacterium specie is Acetobacter xylinum, including the strains ATCC 23769, 10145, 53582, AX5 and many others (Klemm et al 2001). Acetobacter xylinum is an obligate aerobe bacterium usually found in vinegar, alcoholic beverages, fruit juices, fruits, and vegetables, and most likely in rotting ones as well (Klemm et al 2001). The bacteria consume the sugar or carbohydrate from fruits as their main food. BC is formed on the air-liquid medium interface when the HS liquid medium (noted by Hestrin and Schramm, 1954, consisting of 2wt% D-glucose, 0.5wt% peptone, 0.5wt% yeast extract, 0.27wt% disodium phosphate, 0.115wt% citric acid) and distilled water is inoculated with a strain of Acetobacter xylinum. Glucose functions as the bacteria’s carbon source, peptone as a nitrogen source, yeast extract as a vitamin source and citric acid and disodium phosphate as a buffer system for the medium.

The mechanism of BC formation by Acetobacter xylinum is depicted in Figure I-1 and implements as follows (Brown 1996). The bacterium extrudes linear glucan chains from its terminal complexes that are composed of few catalytic sites of extrusion. Approximately 10-100 linear glucan chains aggregate to form into twisting nanofibers. Some papers (Brown, 1976, Zaar 1977) refer to the different level of aggregated glucan chains as sub-elementary microfibrils or microfibrils, but this will be referred to collectively as nanofibers here. The nanofiber from a single bacterial cell with a rectangular cross section of 10-20 x 30-40Å aggregate further to form a ribbon with a diameter of about 70-80 nm. These ribbons are about 20µm long (Zaar 1977). The ribbons are spun into the liquid medium and intertwine with ribbons from other cells to form into a gelatinous suspension or pellicle (Brown 1996). The lateral dimension of BC increases as bacteria grow and as the population increases. During bacterial growth, new production sites for BC become available. Hence, the BC fibril or ribbon widens. At cell division, BC production sites are distributed between two daughter cells, which also increases BC ribbon width (Zaar 1977). BC fibrils aggregate due to hydrogen bonding (Brett 2000) and Van der Waals forces. These forces cause the fibrils to interact, and they are held apart by adsorbed water layers. When the water layers evaporate, the hydroxyl groups of fibril chains associate irreversibly, and a highly crystalline cellulose sheet is formed (Colvin and Leppard 1977). When compared to plant cellulose, BC ribbons are only one one-hundredth in width (Shoda and Sugano 2005). The degree of polymerization (DP) of BC is usually between 2,0 and 6,0 (Jonas and Farah 1998). The morphology of BC depends on the growing culture environment. For a static culture, a leather-like pellicle of overlapping and intertwined ribbons forms (Jonas and Farah 1998). On the other hand, an agitated medium forms irregular BC granules and fibrous strands (Vandamme et al 1998).

(Parte 1 de 4)

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