The Pennsylvania State University

Graduate School

Department of Agricultural and Biological Engineering

EFFECTS OF ADDITIVES ON MECHANICAL AND STRUCTURAL

PROPERTIES ON BACTERIAL CELLULOSE COMPOSITES

A thesis in

Agricultural and Biological Engineering

by

Manmeet Singh Dayal

© 2014 Manmeet Singh Dayal

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Master of Science

August 2014

ii

The thesis of Manmeet Singh Dayal will be reviewed and approved* by the following:

Jeffrey Catchmark

Assistant Professor of Agricultural and Biological Engineering

Thesis Advisor

Virendra Puri

Distinguished Professor of Agricultural and Biological Engineering

Graduate Program chair of Agricultural and Biological Engineering

David Soybel

Nahrwold Professor of Surgery, Penn State Hershey College of Medicine

*Signatures are on file in the Graduate School

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Abstract

Bacterial cellulose (BC) exhibits unique properties including high mechanical

strength and high crystallinity. Improvement in the mechanical properties of BC is sought

for many applications ranging from structural composites to biomedical materials. In this

study, different additives including carboxymethyl cellulose (CMC), pectin, gelatin, corn

starch, and corn steep liquor were added into the fermentation media to alter the BC

produced. Three different concentrations (1%, 3% and 5%) were chosen for each of the

additives, with no additive (0%) being the control. The produced BC was then analyzed

to determine tensile and compression modulus, and a significant increase in these value

were observed (p<0.05). Amongst the tested additives, BC produced in media containing

3% (w/v) pectin had a maximum compressive modulus (142 kPa), and BC produced in

media containing 1% (w/v) gelatin exhibited the maximum tensile modulus (21 MPa).

Pellicle from pectin in media was comparatively harder than control. Substantial variation

in porosity and water holding capacity of BC pellicles were also observed under the

influence of media additives (p<0.05). Structural characteristics of BC and BC-additive

composites were compared using X-Ray diffraction (XRD). The crystal size and

crystallinity of BC was reduced when grown in the presence of CMC and gelatin; of the

additions, pectin only decreased the crystallite size. This suggested that CMC and gelatin

were incorporated into the BC fibril structure. The field emission scanning electron

microscopy (FESEM) images showed the increased micro-fibril aggregation in BC

pellicle on addition of additives to the culture media.

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Table of Contents

List of Figures ................................................................................................................... vii

List of Tables...…………………………………………………………………...……..viii

Acknowledgement ............................................................................................................ iix

Chapter 1: Introduction ....................................................................................................... 1

Chapter 2: Literature review ............................................................................................... 3

2.1 Introduction to cellulose ............................................................................................ 3

2.2 Microbial cellulose .................................................................................................... 4

2.3 Bacterial cellulose ..................................................................................................... 4

2.3.1 Gluconactebacter xylinum .................................................................................. 4

2.3.2 Synthesis of BC............................................................................................. 5

2.3.3 Need for bacterial cellulose .......................................................................... 6

2.3.4 Properties of bacterial cellulose .......................................................................... 8

2.3.5 Bacterial cellulose as a biomaterial .................................................................... 9

2.3.6 Culturing of bacterial cellulose......................................................................... 10

2.2 Additives ................................................................................................................. 12

2.2.1 Pectin ................................................................................................................ 12

2.2.2 Carboxymethyl Cellulose (CMC) ..................................................................... 13

2.2.3 Gelatin .............................................................................................................. 14

2.2.4 Corn Starch ....................................................................................................... 15

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2.2.5 Corn Steep Liquor ............................................................................................ 15

2.3 Sutures ..................................................................................................................... 16

2.3.1 What is a suture? ............................................................................................... 16

2.3.2 Types of sutures and composition of suture material ................................. 16

2.3.3 Suture used in this study ............................................................................. 17

Chapter 3: Goals, Objectives, Hypothesis ........................................................................ 18

Chapter 4: Material and Methods ..................................................................................... 19

4.1 Bacterial strain and stock culture preparation ......................................................... 19

4.2 Cultivation media .................................................................................................... 19

4.3 Inoculum preparation and cultivation ..................................................................... 19

4.4 Additives ................................................................................................................. 20

4.5 Water holding capacity............................................................................................ 20

4.6 Mechanical properties testing.................................................................................. 20

4.6.2 Tension modulus............................................................................................... 20

4.6.1 Compression analysis ....................................................................................... 22

4.7 Density and Porosity ............................................................................................... 23

4.8 BC Crystallinity and crystal size ............................................................................. 24

4.8 BC morphology ....................................................................................................... 25

4.9 Statistical analysis................................................................................................ 26

Chapter 5: Results and discussion..................................................................................... 27

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5.1 Water holding capacity............................................................................................ 27

5.2 Mechanical properties ............................................................................................. 28

5.2.1 Tensile modulus ................................................................................................ 28

5.1.2 Compression modulus ...................................................................................... 29

5.2 Density and porosity................................................................................................ 32

5.3 BC crystallinity and crystal size .............................................................................. 33

5.4 BC morphology………………………………………………….…………….…..37

5.5 General Discussions…………………………………………………….…………39

Chapter 6: General conclusions ........................................................................................ 42

6.1 Conclusion ............................................................................................................... 42

6.2 Suggestions for future work .................................................................................... 43

References ......................................................................................................................... 45

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List of Figures

Figure 1: Structure of single cellulose β-(1,4) linked chain................................................ 4

Figure 2: Biochemical pathway for cellulose synthesis by A. Xylinum CS (cellulose

synthase), GK (glucokinase), PMG (phosphoglucomutase), UGP (pyrophosphorylase

UDPGIc), Glc-6-P (glucose-6-phosphate), UDPGIc (uridine diphosphoglucose), Glc-1-P

(glucose-1-phosphate) (Ross et al., 1991)........................................................................... 6

Figure 3: A typical structure of Pectin .............................................................................. 13

Figure 4: A typical structure of CMC ............................................................................... 14

Figure 5: A typical structure of Gelatin (Liu, Liu, & Wang, 2010) .................................. 15

Figure 6: a) Dynamic mechanic analyzer (model Q800, New Castle, DE); b)Sample

mounted between the two clamps. .................................................................................... 21

Figure 7: a) Intron 3375 (Norwood, MA); b) Mounting of the BC sample; c) Initial

compression the BC sample; d) Compressed BC sample and water oozing out. ............. 23

Figure 8: a) D/Max Rapid II diffractometer (Tokyo, Japan); b) Setting up sample for

analysis. ............................................................................................................................. 25

Figure 9: Nova NanoSEM 600 (Czech Republic, Europe) ............................................... 26

Figure 10: Tensile stress-strain curves for BC pellicles. .................................................. 29

Figure 11: Compressive stress-strain curves for BC pellicles. ......................................... 31

Figure 12: Typical XRD curves of BC (Park et al., 2010). .............................................. 34

Figure 13:X-ray diffraction patterns of BC and BC-composites. ..................................... 36

Figure 14: FESEM images of BC pellicles at different magnifications. a), b), c) BC

pellicle; d), e), f) BC-CMC 1% Pellicle; g), h), i) BC-Pectin 1%; j), k), l) BC-Pectin 3%;

m), n), o) BC-Gelatin 1% .................................................................................................. 38

viii

List of Tables

Table 1: Mechanical properties and Water holding capacity of BC-composites (Mean ±

Standard deviation) ........................................................................................................... 31

Table 2: Density and porosity of the selected BC pellicles .............................................. 33

Table 3: Structural features of BC/composites ................................................................. 36

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Acknowledgement

I would want to extend my profound gratitude and regards to Dr. Jeffrey

Catchmark, who was abundantly helpful and offered invaluable support and supervision

throughout the course of this thesis. His impeccable guidance, enthusiasm and patience

added to my graduate experience and helped me work efficiently. His well-equipped

laboratory and lively nature was a big motivation in this research. I am very grateful to

receive his constructive criticisms during the course.

I would take this opportunity to thank Dr. Virendra Puri for his cordial support,

and valuable guidance, which helped me compiling this work more logically and

quantitatively. Also, I would like to thank Dr. David Soybel for taking time out from his

busy schedule to serve as committee memeber.

Very special thanks go out to the technicians at Materials Research Institute, The

Pennsylvania State University. I acknowledge their kindness and technical assistance

while working in their laboratory.

I must acknowledge the support I received from my lab mates, Niharika Mishra,

Jin Gu, Lin Fang, Yuanyuan Weng, Adam Plucinski, Yuzhi Deng, Snehasish Basu, and

Kai Chi. It was a wonderful time for our exchanges of knowledge and lab lunches, which

helped enrich the experience. I am obliged to faculty, staff members, and colleagues of

ABE department for cooperation provided by them in their respective fields during the

period of my Masters.

Lastly, I want to express my gratitude for my parents, sister Garima and friends,

to name a few, Nitin, Radhika, Raghu, Jyotsna, Omkar, Kandhar, Nitish, and Rahul, for

their support, encouragement and love all through these two years of the course.

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Chapter 1

Introduction

Cellulose is a polymer of glucose units linked together by β-(1–4)-glycosidic

linkages, and is the most abundant renewable resource on Earth. It is a very important

and fascinating biopolymer and almost inexhaustible raw material. The tailoring of

innovative products for science, medicine and technology has led to global regeneration

of interdisciplinary cellulose research and to the extended use of this abundant polymer

over the last decade (Ciolacu & Popa, 2011). Apart from plants, many strains of bacteria

possess the ability to produce cellulose (Gea et al., 2011a) . Plant cellulose (PC) is

obtained with various impurities such as lignin and hemicellulose and thus requires harsh

chemical treatments to remove these impurities, which results in irreversible changes in

cellulose structure and thus strip the polymer of its useful characteristics (Sheykhnazari,

Tabarsa, Ashori, Shakeri, & Golalipour, 2011) whereas bacterial cellulose (BC) is free of

such impurities and is obtained as highly pure cellulose. BC does not require remedial

processing to remove unwanted polymers and contaminants and therefore, retains a

greater degree of polymerization (Nishi et.al., 1990). This fact gives bacterial cellulose

superior unidirectional strength.

Adding different additives into the culture media enhanced BC production.

Calcofluor White ST, an optical brightener (stain) for cellulose, altered the rate of

glucose polymerization into cellulose and disrupted the assembly of crystalline cellulose

1 when the concentration was increased above 0.1mM (Benziman et al., 1980). Addition

of other polysaccharides such as pectin, carboxymethylcellulose (CMC), sodium alginate,

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and corn steep liquor were reported to enhance BC production (Chen et al, 2009; Ishida et

al., 2003; Gu et al., 2013).

The nano-scale morphology shows the presence of nano-fibrils; however, it is

more likely the structure of the entire network at a micro- level that can explain the

extremely good mechanical properties of Bacterial Cellulose. The mechanical properties

of BC pellicle have been investigated. According to Yamanaka et al. (2009), BC sheets

gave a Young’s modulus of around 15 GPa.

Copious amount of research is done on enhancing BC production using different

additives, but a comprehensive study of these BC products and their mechanical and

structural properties is sparse.

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Chapter 2

Literature review

This literature review will provide a background on the state of the art in microbial

cellulose production for biomedical applications and set the foundation for the proposed

experiments.

2.1 Introduction to cellulose

A popular naturally occurring polymer, cellulose, is the world’s most abundant,

renewable and biodegradable polymer. Systematic study of cellulose started after it

was discovered by the French agricultural chemist Anselme Payen in 1834. It is an

organic compound with hundreds or thousands of glucose units linked together by β-

(1–4)-glycosidic bonds, and for a straight chain (Figure 1). The hydroxyl groups on

the glucose from one chain form hydrogen bonds with hydroxyl group on an adjacent

chain, bundling the chain together. Cellulose is an important structural component of

cell wall of higher plants and is an almost inexhaustible raw material. The crafting of

innovative products for agriculture, food, medicine and technology has led to global

reinforcement in the interdisciplinary research on cellulose and to the applications of

this profuse polymer over the past few years.

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Figure 1: Structure of single cellulose β-(1,4) linked chain

2.2 Microbial cellulose

Cellulose is an important industrial material and to meet the demand cellulose

using a microbial system was the alternate source. Apart from plants, different types

of microorganisms possess the ability to produce cellulose (Gea et al., 2011a).

Microbial cellulose can be produced from the microorganisms such as fungi

(Saprolegnia sp., Dictystelium discoideum), algae (Vallonia sp.), bacteria

(Acetobacter sp., Aerobacter sp., Agrobacterium sp., Azotobacter sp., Pseudomonas

sp., Rhizobium sp., Sarcina sp., Salmonella sp., Zoogloea sp.), amoebae, cellular

slime molds and green algae (Gea et al., 2011a; Sun, Zhou, Wu, & Yang, 2007).

Amongst different microbial strains reported for cellulose production

Gluconactebacter is one of the most widely studied strains and is commercially

exploited for cellulose production (Gea et al., 2011a).

2.3 Bacterial cellulose

2.3.1 Gluconactebacter xylinum

The gram-negative rod shaped bacterium Gluconactebacter xylinum is

regarded as the most efficient producer of bacterial cellulose (Setyawati, Chien, &

Lee, 2007). A cell of Gluconactebacter xylinum can polymerize up to 200,000

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glucose molecules per second into β-1,4-glucan chains which are forced out into the

surrounding medium in the form of a single, twisted, ribbon like bundle of

microfibrils (Ross, Mayer, & Benziman, 1991). Because of its ability to produce

relatively high levels of cellulose, G. xylinus serves as a model system to study

cellulose synthesis. Because of its ability to produce relatively high levels of

cellulose, G. xylinus serves as a model system to study BC synthesis.

2.3.2 Synthesis of BC

The bacteria synthesize cellulose as a primary product. The mechanism of

cellulose microfibril formation has also been investigated actively .The bacteria

synthesize cellulose as a primary product. Bacterial Cellulose (BC) is synthesized in

three stages. In first stage, glucose molecules are polymerized by the formation of

glucosidic linkages between the outer and cytoplasm membranes, forming cellulose

chains. 10-15 of such parallel chains form a 1.5nm wide protofibril. In the second

step several protofibrils condense to into 2-4 nm wide microfibrils. In the third step

a bundle of microfibrils are assembled into a 20-100 nm wide ribbon (Castro et al.,

2011). The ultrafine BC ribbons (with lengths ranging from 1 to 9 μm) form a dense

reticulated layer by layer structure, which is stabilized by extensive hydrogen

bonding (Chen, P., S.Y. Cho, 2010) .

The biosynthesis of bacterial cellulose is a multifold stepped process that

involves two main mechanisms; the synthesis of the cellulose precursor uridine

diphosphate glucose (UDP-GIc), followed by the polymerization of glucose into

long and unbranched chains (the β-1-4 glucan chain). The carbon compounds

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undergo conversion to hexoses via gluconeogenesis (Ross et al., 1991). The hexoses

then goes through phosphorylation to glucose-6-phosphate (Glc-6-P), catalyzed by

glucokinase, followed by isomerization of this intermediate to glucose-1-phosphate

(Glc-1-P), catalyzed by phosphoglucomutase, and conversion of the latter metabolite

to UDP-Glc by UDP-Glc-pyrophosphorylase. This is followed by glucose

polymerization into the β-(1-4)glucan chain and a nascent chain which forms

ribbon-like structure of cellulose chains formed by hundreds or even thousands of

individual cellulose chains (Ross et al., 1991; Vandamme, De Baets, Vanbaelen,

Joris, & De Wulf, 1998).

Figure 2: Biochemical pathway for cellulose synthesis by A. Xylinum CS (cellulose

synthase), GK (glucokinase), PMG (phosphoglucomutase), UGP (pyrophosphorylase

UDPGIc), Glc-6-P (glucose-6-phosphate), UDPGIc (uridine diphosphoglucose), Glc-1-P

(glucose-1-phosphate) (Ross et al., 1991)

2.3.3 Need for bacterial cellulose

Plant cellulose (PC) is obtained with various impurities such as lignin and

hemicellulose and thus requires harsh chemical treatments to remove these

impurities, which results in irreversible changes in cellulose structure and thus

strip the polymer of its useful characteristics (Sheykhnazari et al., 2011). Whereas

7

bacterial cellulose (BC) is free of such impurities and is obtained as highly pure

cellulose. BC does not require remedial processing to remove unwanted polymers

and contaminants and therefore, retains a greater degree of polymerization (Nishi

et.al., 1990). This fact gives bacterial cellulose superior unidirectional strength.

Both plant cellulose (PC) and bacterial cellulose (BC) have the same

molecular formula, (C6H10O5)n but they differ in their highly ordered structure

(Makoto Shoda, 2005), and in their physical and chemical properties (Gea et al.,

2011b). Plant cellulose (PC) is obtained with various impurities such as

hemicellulose and lignin and thus harsh chemical treatments to remove these

impurities are required. This results in irreversible changes in cellulose structure

and thus strip the polymer of its useful characteristics (Sheykhnazari et al., 2011).

Bacterial cellulose is free of such impurities and is obtained as highly pure

cellulose. BC does not require remedial processing to remove unwanted polymers

and contaminants and therefore, retains a greater degree of polymerization

(Division, 1989). Furthermore, some issues currently exist in the production of

plant cellulose products. These problems include pollution, which occurs during

the pulping and bleaching, processes and large amounts of liquid waste and toxins

that are discharged from cellulose even without any form of treatment. Costly

treatments are required in order to reduce these harmful effects (Chen, P., S.Y.

Cho, 2010). BC does not require remedial processing to remove unwanted

polymers and contaminants and therefore, retains a greater degree of

polymerization (Division, 1989). BC is secreted in the form of a ribbon, composed

of bundles of microfibrils (Ross et al., 1991) which gives bacterial cellulose a

8

superior unidirectional strength. BC is one of the most promising biological

materials due to its unique fibrillar nanostructure, giving it advantages over PC in

terms of physical and chemical properties.

2.3.4 Properties of bacterial cellulose

Due to the super molecular structure of BC, it has distinguished physical and

chemical properties. BC has a higher crystallinity with values of 60-90% and due to

absence of any impurity it possesses higher purity (Makoto Shoda, 2005) It has an

improved water holding capacity (Tang, Jia, Jia, & Yang, 2009), and increased

tensile modulus (Publishers, 2000), porosity, and moldability. Beyond the structural

and mechanical properties, BC also exhibits a good biodegradability and good

biocompatibility (Wei, Yang, & Hong, 2011). Additionally, the choice of a

cultivation technique also influences the physical and mechanical properties of the

microbial cellulose. In stationary culture conditions, a thick, gelatinous membrane of

BC is accumulated on the surface of a culture medium, whereas under agitated

culture conditions, cellulose can be produced in the form of a fibrous suspension,

irregular masses or spheres (Krystynowicz et al., 2002). These exceptional

properties of BC make it a very important raw material in various commercially-

important industries such as food, textile, paper, and medicine, for producing

composite membranes, biomedical material, bio-sorbent material, loud speaker

diaphragms, and dessert foods material (Makoto Shoda, 2005) , and in wound

healing and as scaffolds material (Yang et al., 2011).

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2.3.5 Bacterial cellulose as a biomaterial

Bacterial cellulose has become to be an extraordinarily adaptable biomaterial

and thus can be used in a wide variety of applied scientific research activities, such

as paper products, agriculture, electronics, food, pharmaceutical drug carrier,

acoustics, and biomedical devices. The research in biomedical devices has recently

multiplied by a significant amount due to an increased interest in tissue-engineered

products for both wound care and the regeneration of damaged organs. The unique

nanostructure and properties of bacterial cellulose has consequently made BC a

natural candidate for numerous medical and tissue-engineered applications (Czaja et

al., 2006). One of the major requirements of any biomedical material is that it must

be biocompatible, which is the ability to remain in contact with living tissue without

causing any toxic or allergic side effects. BC consists of completely pure cellulose

nanofibrils and induces minor foreign body and inflammatory responses; thus it is

considered as biocompatible (Helenius et al., 2005). It is desirable to utilize a

biomaterial that has the porosity required to support cell ingrowth and effective mass

transport (Svensson et al., 2005); microbial cellulose is a highly porous material

with pore sizes ranging from several nanometers to micrometers in size.

The BC fibers have a high aspect ratio with a diameter of 20-100 nm and

thus have a very high surface area per unit mass. This property, combined with its

highly hydrophilic nature, results in a very high liquid loading capacity which can be

utilized to maintain a moist environment at the wound dressing surface (Rezai,

2011). BC possesses not only great water holding ability and mechanical properties,

but also excellent biodegradability (Miyamoto et al., 1989). The high elasticity and

10

the ability to be molded in situ of BC helps in the fabrication of different shapes and

sizes of biomaterial that provide easy and close wound coverage. The nonwoven

ribbons of bacterial cellulose microfibrils closely resemble the structure of native

extracellullar matrices, suggesting that BC could function as a scaffold for the

production of many tissue-engineered constructs (Czaja et al., 2006). The use of

biomaterial in tissue engineering is essential in order to support cell proliferation and

maintain their differentiated functions. According to Liuyun et al. (2009) the cells

attached and proliferated on a biomaterial made from microbial cellulose thereby

indicating that the biomaterial was non-toxic and had good cell biocompatibility.

2.3.6 Culturing of bacterial cellulose

The fermentation medium contains carbon, nitrogen and other macro- and

micronutrients required for the growth of organisms. The changes in the medium

components affect the growth and the product formation directly or indirectly.

Usually, glucose and sucrose are used as carbon sources for cellulose production,

although other carbohydrates such as fructose, maltose, xylose, and glycerol have

also been tried (Keshk and Sameshima, 2005). The cellulose yield is related to the

amount of glucose that is consumed (Chen, P., S.Y. Cho, 2010). Cellulose

production requires a specific complex nitrogen source, which is a main

component of proteins necessary in cell metabolism, and provides not only amino

acids but also vitamins and mineral salts (Coban and Biyik, 2011). The preferred

nitrogen sources are yeast extract and peptone. Also, corn steep liquor had a

stimulating effect on cellulose production (Tang et al., 2009).

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Temperature is a vital factor that affects growth of bacteria and cellulose

production. The maximal cellulose production was observed between 28-30°C.

Temperature variations cause changes in the polymerization pattern of cellulose

fibers and the water-binding capacity of cellulose (Chawla, Bajaj, Survase, &

Singhal, 2009). pH of the culture medium is also an important factor that affects

the cell growth and cellulose production. The optimum pH of the culture medium

for bacterial cellulose production is in the range of 4.0 to 6.0 (Chawla et al.,

2009).

In static cultures the cellulose pellicle is formed on the interface of the

medium and air. Thus the oxygen available for cell metabolism might become

limiting and could have a negative influence on cellulose production (A Shirai, M

Takahashi, H Kaneko, S Nishimura, M Ogawa, N Nishi, 1994) .In the static

culture conditions a thick, gelatinous cellulosic film is accumulated on the

interface of medium and air of the culture medium (Ross et al., 1991), whereas

under agitated culture conditions cellulose can be produced in the form of a

fibrous suspension, irregular masses or spheres. It has been reported that cellulose

production is much less under agitated culture conditions than in static culture

(Chen, P., S.Y. Cho, 2010). A two-stage fermentation process, comprising

agitated culture for propagation and static culture for pellicle formation has also

been reported (Okiyamaet al., 1992).

The effect of inoculation volume and culture time on thickness of bacterial

cellulose membranes has been observed (Tang et al., 2009). With the increase in

cultivation time, the yield of bacterial cellulose and cell mass increased due to

12

accumulation of more fibrils. The bacterial strain is usually cultivated in a

standard medium suggested by Hestrin and Schramm (HS) (Schramm & Hestrin,

1954) consisting of (w/v): glucose (2.0%), peptone (0.5%), yeast extract (0.5%),

disodium phosphate (0.5%) and citric acid (0.15%).

2.2 Additives

2.2.1 Pectin

Pectin, a structural heteropolysaccharide, is a gel-forming component in

primary cell walls of higher plants (Albersheim et al., 2010). This sugar polymer

is known to provide strength to the cell wall and assist cell to cell adhesion. Both,

cellulose and pectin are present in primary cell walls yet very little information is

available on their interaction with each other. Pectin can be extracted from fruits

such as apple, grapes, apricots, and pears and also from citrus peel. Pectin is

known to have acomplex structure with primary molecule of galacturonic acid

linked to each other with α- (1,4)-glycosidic bonds (Figure 4 ). It forms

independent networks although it is localized close to cellulose ribbons (Tokoh,

Takabe, Sugiyama, & Fujita, 2002).

Its networks are phase separated with the dense cellulose microfibrils.

According to Gu et al (Gu & Catchmark, 2012) pectin and cellulose may interact

with each other at different points during the assembly process or even in

different regions. Pectin impacts the structure and degree of crystallinity when

present in the BC culture medium and stimulates the growth of cellulose

(Szymańska-Chargot, Cybulska, & Zdunek, 2011).

13

Figure 3: A typical structure of Pectin

2.2.2 Carboxymethyl Cellulose (CMC)

CMC is a derivative of cellulose. It has similar structure as of cellulose

with carboxymethyl groups attached to some of the hydroxyl groups of β-(1,4)-

glucose polymer of cellulose (figure 5). The carboxyl group makes it soluble in

water. CMC is classified by the US FDA as generally recognized as safe (GRAS)

and can be used as a food additive. (Chaplin, n.d.)

CMC interacts with cellulose fibers and alters the regularity of its fibril

structure. With the addition of CMC, the BC production increases possibly due to

the presence of the additional carbon. Crystallization is known to be a limiting

step in cellulose production. CMC participates in hydrogen bonding with the BC

micro-fibrils during fermentation and is observed to decrease the crystallinity of

BC samples (Yu, 2011). The CMC has an inter-penetrating network with BC

which made the cellulose ribbons larger and denser (Chen, Chen, Huang, & Lin,

2011) . According to Hayyim and Ohad (Ben-Hayyim & Ohad, 1965) the CMC

14

incorporated cellulose consists of crossed and superimposed layers of parallel

oriented cellulose fibrils.

Figure 4: A typical structure of CMC

2.2.3 Gelatin

Gelatin is a colorless and flavorless water-soluble protein derived from

hydrolysis of collagen. A typical structure of gelatin is shown if Figure 6. It

possesses properties of an ideal biomaterial for many in-vivo healthcare

applications, i.e., good biocompatibility, low immunogenicity, and

biodegradability (Wang et al., 2012). Due to these properties gelatin is clinically

proven as a wound dressing material (Kim, Yi, Choi, Kim, & Lee, 2012).

It forms a firm gel at ambient temperatures and is used in food as a gelling

agent. According to Chang et al. (2012) hydrogen bonds are major conjugate

forces between BC and gelatin. The BC forms a mesh like structure and gelatin

fills the network (Chang, Chen, Lin, & Chen, 2012).

15

Figure 5: A typical structure of Gelatin (Liu, Liu, & Wang, 2010)

2.2.4 Corn Starch

Corn Starch is a fine, powdery starch derived from the endosperm of corn

kernels. It is used as a thickening agent and has many culinary uses. Corn starch

does not alter the ribbon assembly in BC (Haigler, White, Brown, & Cooper,

1982). Grande et al. (2009) speculates that Acetobacter bacteria do not consume

the starch and that it remains present in the final BC nano-composite (Grande et

al., 2009).

2.2.5 Corn Steep Liquor

Corn steep liquor (CSL) is a viscous concentrate obtained as by-product of

corn wet-milling and has been used as a nitrogen supplement in several different

fermentation media. CSL being rich in proteins, vitamins, inorganic ions, and

myo-insitol phosphates is more effectively used by the microorganism than yeast

extract and peptone for the production of BC (Houssni El-Saied, Ahmed I. El-

Diwany, Altaf H. Basta, Nagwa A. Atwa, 2008). CSL has lactate that has a

16

metabolic effect on the bacteria. It induces high cell growth and cellulose

production in comparison to the other nitrogen sources (Jonas & Farah, 1998).

2.3 Sutures

2.3.1 What is a suture?

A suture is a therapeutic device used to grip body tissues together after a

surgery or a wound. Suture is a fiber strand with a metallic needle attached at one

of its ends and is necessary in almost every surgical operation (Manavalan &

Mukhopadhyay, 2009). The objectives of a suture are closing, supporting and

strengthening wounds until healing Gut sutures in modern day are made of

specially prepared beef and sheep intestine.

2.3.2 Types of sutures and composition of suture material

There are various types of sutures available. Sutures can be divided into

two types – absorbable or non-absorbable. Absorbable sutures, as the name

suggests are absorbed by the body cells and tissue fluids and will break down,

without causing harm, in the body over time. The most commonly used suture

absorbable suture material is the surgical gut, which is derived from the small

intestine of healthy sheep. Another type of absorbable suture, chromic sutures, has

undergone various concentrations of tanning with the salt of chromic acid. Non-

absorbable sutures are made of special silk or the synthetics polypropylene and

are not absorbed by the body and must be manually removed if they are not left

indefinitely. Nylon and polymer polypropylene are most commonly used non

17

absorbable suture materials. Sutures can also be divided into two types on the

basis of material structure i.e. monofilament sutures and multifilament or braided

sutures (Glenn N. Wagnera, 2000).

2.3.3 Suture used in this study

PROLENE Polypropylene Suture from ETHICON ( a subsidiary of Johnson

& Johnson) is used in this study. It is a non-absorbable mono filament suture with

a diameter of 0.2 mm. For easy identification of these sutures during a surgery

they are coloured blue. It is used for skin closure and general soft tissue closure

(Http://www.ethicon.com/healthcare-professionals/products/wound-closure/non-

absorbable-sutures/prolene-polypropylene#!description-and-specs, n.d.).

18

Chapter 3

Goals, Objectives, Hypothesis

The unique properties associated with its super-molecular structure, as stated in

the literature review, have made bacterial cellulose a fascinating and a versatile material

for industrial applications.

The goal of this research is to compare the mechanical and structural properties of

BC grown in presence of different additives with three different concentrations.

The specific objectives leading to accomplishment of this goal are:

To study the influence of different additives in varying concentrations on

compression modulus and tensile modulus.

To determine the best combinations of additive and its concentrations for high

mechanical properties of BC.

To compare the structural differences of BC composites by evaluating the

crystallinity, using X-Ray diffraction (XRD), and Polymer arrangement using

field emission scanning electron microscope (FESEM).

Hypothesis

Addition of additives to the growth media will result in a BC pellicle with

better mechanical properties than the control.

19

Chapter 4

Material and Methods

4.1 Bacterial strain and stock culture preparation

Gluconacetobacter xylinus 53582 used in this study was obtained from American

Type Culture Collection (Rockville, MD). The cell suspension of the bacteria was stored

at -80C in a 20% glycerol solution.

4.2 Cultivation media

All chemicals used were of analytical grade and commercially available, unless

specified. The BC/BC-composites production was studied in Hestrin-Schramm medium

(1954) consisting of (per liter): 20.0 g of glucose, 5.0 g of yeast extract, 5.0 g of peptone

and 2.7 g of disodiumhydrogen phosphate, 1.5 g of citric acid, and 1.0 g of magnesium

chloride.

4.3 Inoculum preparation and cultivation

The frozen cell stock culture was thawed and added to 100mL of Hestrin-

Schramm (HS) medium in a 250mL Erlenmeyer flask for cell culture revival. The flask

was kept at 30C for 3 days. The culture grown in this medium was used as inoculum for

further studies. The BC production studies were performed in 100 ml culture media,

contained in a 250ml Erlenmeyer flask, inoculated with 1mL cell suspension at 30C for

14 days under static conditions. The additives were added to the media in these flasks

prior to inoculation.

20

4.4 Additives

This study includes the effects of different concentrations of different additives.

The additives used were carboxymethyl cellulose, pectin, gelation, corn steep liquor, and

corn starch, each with a concentration of 1%, 3% and 5%. For every analysis 3 replicates

were taken.

4.5 Water holding capacity

The alkaline washed BC-composite pellicles were removed from the flask. The

pellicle was shaken twice and placed horizontally onto a stainless steel mesh to allow the

water to drain. In order to avoid evaporation, the pellicles were covered. The wet weight

of the pellicle was measured after 5 minutes.

The samples were dried at 30ºC for 12 hours. To minimize rehydration each

pellicle was quickly transferred to the weighing balance and the dry weight was taken.

The water holding capacity (WHC) was calculated by using the following formula:

The mean WHC and its standard deviation were calculated based on three samples.

4.6 Mechanical properties testing

4.6.2 Tension modulus

The Young’s modulus of BC-composites was calculated from the stress-

strain experiments performed by a dynamic mechanic analyzer (DMA) (model

Q800, TA instrument-Water LLC, New Castle, DE) (Figure 7). The alkaline

washed BC-composites were pressed into a flat piece at 25 Newton using Instron

21

model 3345. The BC-composite samples were then cut to make 20 × 3-4 mm

(length × width) plates and the thickness was measured prior to loading the

samples for testing. The samples were then mounted between fixed (upper) and

movable (lower) clamps, and two ends were fixed to avoid slip. Force was applied

to the lower clamp to pull the sample in tension. Experiments were run at a strain

rate of 1%/min to a final strain value of 70%. Tests were performed at room

temperature (approximately 25°C). Stress (σ) was calculated by F/A where F is

loading force in Newton (N) and A is the cross-section area measured as width ×

thickness of sample. Strain (ε) was calculated by ΔL/Lo where Lo is the initial

length and ΔL is sample extension. Young's modulus was calculated by

stress/strain in the first linear region of the graph. The mean Young’s modulus

and its standard deviation were calculated based on three samples.

Figure 6: a) Dynamic mechanic analyzer (model Q800, New Castle, DE); b)Sample

mounted between the two clamps.

22

4.6.1 Compression analysis

The Young’s modulus in compression was calculated using and Instron

model 3345 (Norwood, MA) (Figure 8). A 20 mm 20mm sample from each

pellicle was taken and compressed to a force of 25 Newton. Sample thickness was

measured before compression. Experiments were run at a displacement rate of

1.5mm/minute. Tests were performed at room temperature (approximately 25°C).

Stress (σ) was calculated by F/A where F is loading force in Newtons (N) and A

is the cross-section area measured as width × thickness of sample. Strain (ε) was

calculated by ΔL/Lo where Lo is the initial length and ΔL is exerted compression

from the starting point. Young's modulus was calculated by stress/strain in the

linear region. Figure 1 shows a stress-strain response of the control (BC) and BC-

composites. The mean Young’s modulus and its standard deviation were

calculated based on three samples.

23

Figure 7: a) Intron 3375 (Norwood, MA); b) Mounting of the BC sample; c) Initial

compression the BC sample; d) Compressed BC sample and water oozing out.

4.7 Density and Porosity

A 2020 mm sample was cut using a scissor from every BC pellicle and its

thickness was recorded. Each sample was kept on a mesh for 5 minutes to drain the

surface water and then the sample was weighed. Density was calculated using the

formula:

24

The porosity of BC films has been calculated from the measured density using the

following formula:

( )

where ρs corresponds to the density of the sample and ρc is the absolute density for the

cellulose (1.5 g/cm3) (Retegi et al, 2011).

4.8 BC Crystallinity and crystal size

X-ray diffraction data, used to study the crystallinity of the composites, were

recorded using a D/MAX Rapid II diffractometer (Shibuya-Ku ,Tokyo) (Figure 9). The

samples were freeze-dried for XRD analysis. The operating voltage and current were

50kV and 40 mA, respectively. The samples were exposed for a period of 5 minutes

using Cu radiation with a wavelength of 0.154 nm.

Peak Fit software (Livermore, California) was used to extract individual peak

information. The results from a fit included a total profile, crystallinity, d-spacing, and

crystal size. A good fit was obtained when five peaks were selected for the fitting. Four

of the peaks were associated with the characteristic cellulose I, and the fifth peak at

around 21.5º was assigned to the amorphous fraction. The crystallinity index was

calculated using the peak deconvolution method (Park, Baker, Himmel, Parilla, &

Johnson, 2010). According to this method the software uses a curve-fitting process that

separates the amorphous and crystalline contributions to the diffractrogram. The

crystallinity is calculated as the ratio of the area of all crystalline peaks to the total area

(both amorphous and crystalline).

25

Figure 8: a) D/Max Rapid II diffractometer (Tokyo, Japan); b) Setting up sample for

analysis.

4.8 BC morphology

To observe microstructure of the BC-composites field emission scanning electron

microscopy (FESEM) was conducted. Freeze-dried samples were sputter coated with

gold. NanoSEM 600 field emission scanning electron microscope (Czech Republic,

Europe) (Figure 10) at 10 kV was used for sample examination.

26

Figure 9: Nova NanoSEM 600 (Czech Republic, Europe)

4.9 Statistical analysis

Minitab software (Version 16.2, State College, PA) was used to perform t-test

analysis on the data; determining whether the relationships of additives and the BC

properties are significant or not. The hypothesis was tested at 95% confidence level using

Minitab software.

27

Chapter 5

Results and discussion

The mechanical and morphological properties of BC and its composites are

presented in this chapter. The thickness of BC pellicles varied across composites due to

presence of additives. The thickness was recorded in Table 1. The composites of

BC/CMC-1%, BC/P-1%, BC/P-3%, and BC/G-1% were selected to compare to control

BC for further morphological and structural studies as they exhibited the best mechanical

properties. In addition, the comparisons of the additives’ effect on different properties are

discussed. Furthermore, the relationships of the additive and its concentration with the

mechanical and structural properties of each BC are developed and validated in the

subsequent sections.

5.1 Water holding capacity

The wet weight and the dry weight were measured to calculate the water holding

capacity. The effect of the additives and their varying concentration on WHC of BC-

composites is presented in Table 1. The control (BC) significantly has a higher WHC as

compared to the other BC-composite pellicles (p<0.05). However, a trend is observed

within the different concentrations of an additive. The WHC decreases considerably with

an increasing concentration of the additive (p<0.05). This suggests that the void spaces in

the BC structure are filled with the additive and the water is pushed out. Similar

observations were previously reported by Taokaew and his research group (Taokaew,

Seetabhawang, Siripong, & Phisalaphong, 2013).

28

5.2 Mechanical properties

5.2.1 Tensile modulus

Figure 11 shows the stress-strain curves of four BC-composite, as stated

initially, with the control BC pellicle under uniaxial tensile test. As clearly visible

in the graph, the modulus of the curves increases with the addition of additives in

their respective concentrations.

The value of the tensile modulus of the control BC is consistent to the

earlier studies with uniaxial mechanical tests, reporting values between 1-10 MPa

(McKenna, Mikkelsen, Wehr, Gidley, & Menzies, 2009). The effect of the

additives and their varying concentration on Young’s modulus of BC-composites

is presented in Table 1. The Young’s modulus of the BC-composites increased

with the addition of the additives. This behavior could be due to the formation of

interactions between additive and BC. The BC fibril structure was entangled with

the presence of the additives in the growth media. This gave a more compact and

rigid chemical structure to the BC pellicle (Figure 14). As a result, the Young’s

modulus of the BC/composite increased. Gelatin and CMC are known to form

hydrogen bond with BC during its aggregation phase (Haigler et al., 1982;

Taokaew et al., 2013). Whereas pectin does not chemically react with BC but is

present in the vicinity of BC fibers and is entangled with these fibers.

Both the additive and their respective concentrations (1%, 3% and 5%)

had a significant effect on the tensile modulus of certain BC pellicles (p<0.05),

but not on all samples (p>0.05). When compared to the control, there is a

significant increase in tensile modulus of the BC composites with 1% (w/v) pectin

29

and 1% (w/v) gelatin (p<0.05) in the culture media. As reported in Table 1, the

BC/Gelatin-1% composite gave the statistically highest (p<0.05) mean value of

modulus of about 21.94 MPa.

Figure 10: Tensile stress-strain curves for BC pellicles.

5.1.2 Compression modulus

The effect of the additives and their varying concentration on compression

properties of BC-composites is presented in Table 1. The fibril bundling and

orientation along with the degree of interaction between micro-fibrils in BC has a

major influence on the mechanical properties (Henriksson et al. 2008). Figure 12

shows the stress-strain curves of the four BC-composite, as stated initially, with

the control BC under compression force. The stress-strain curves exhibit an initial

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0.01 0.06 0.11 0.16 0.21 0.26 0.31 0.36

BC

BC/CMC 1%

BC/G 1%

BC/P 1%

BC/P 3%

Stain (mm/mm)

Strs

s (M

Pa)

30

linear region in all cases. The slope of this linear region was the Young’s modulus

of the composites. With the increase in load the cellulose structure starts to

collapse, giving a stress plateau. The non-linear curve is obtained because of the

breakdown within the network of fibers during deformation. BC is well known to

exhibit a layered structure. When the layers of BC within the pellicle come in

contact with each other under compression, there is a steep increase in stress due

to densification.

The enhancement in compression properties observed is associated with

the interaction between the BC and the additive forming a stiffer network

structure. Pectin is a well-known gelling agent used in food manufacturing. The

incorporation of pectin into the BC matrix may be responsible for the improved

compressive modulus even if there is little interaction between pectin and

cellulose.

Both the additive and their respective concentrations (1%, 3% and 5%)

had a significant effect on the compressive modulus (p<0.05). As reported in

Table 1, the BC/Pectin-3% composite gave the statistically highest (p<0.05) mean

value of modulus of about 142.3 kPa. Since the additives are incorporated within

the BC structure and the pellicle formed tends to resist the force applied, it does

change the compressive modulus significantly. The compression of samples did

not ooze out the cellulose; the water in the pellicle was forced out. This was

confirmed using Calcofluor White staining on the water forced out. The stain is a

non-specific fluorochrome and binds to cellulose, giving it fluorescence and

diminished the background fluorescence.

31

Figure 11: Compressive stress-strain curves for BC pellicles.

Table 1: Mechanical properties and Water holding capacity of BC-composites (Mean ±

Standard deviation)

SAMPLE

Tensile Modulus

(MPa)

Compressive

Modulus (kPa)

Water holding

capacity

BC (Control) 7.55 ± 0.33 7.43 ± 0.70 32.34 ±8.65

BC + CMC 1% 1 5.93 ± 1.03 107.75 ± 5.16 18.20 ±3.08

BC + CMC 3% 4.03 ± 0.17 21.60 ± 6.79 13.11 ± 4.39

BC + CMC 5% 8.45 ± 2.17 25.55 ± 4.88 11.49 ± 4.17

1 CMC = Carboxymethyl cellulose,

0

0.05

0.1

0.15

0.2

0.25

0 0.2 0.4 0.6 0.8 1

BC

BC/P-1%

BC/G-5%

BC/P-3%

BC/CMC-1%

Str

ess

(kP

a)

Strain (mm/mm)

32

BC + P 1% 2 16.70 ± 1.91 80.03 ± 4.49 22.99 ± 0.64

BC + P 3% 9.09 ± 3.60 142.30 ± 2.12 17.23 ± 4.74

BC + P 5% 5.06 ± 1.03 129.27 ± 8.43 12.33 ± 3.26

BC + G 1% 3 21.94 ± 1.70 83.05 ± 2.19 23.23 ± 1.18

BC + G 3% 3.67 ± 1.92 80.40 ± 9.33 15.46 ± 5.78

BC + G 5% 7.70 ± 2.01 60.75 ± 5.73 10.14 ± 0.66

BC + CSL 1% 4 8.54 ± 0.69 26.70 ± 2.81 25.27 ± 1.56

BC + CSL 3% 6.05 ± 1.37 46.10 ± 6.64 22.25 ± 5.97

BC + CSL 5% 5.62 ± 0.48 46.87 ± 4.07 14.39 ± 1.58

BC + CS 1%5 3.84 ± 1.01 81.58 ± 5.40 40.94 ± 1.35

BC + CS 3% 3.24 ± 1.60 82.24 ± 4.77 29.97 ± 4.79

BC + CS 5% 3.44 ± 0.93 87.10 ± 1.84 18.60 ± 0.13

5.2 Density and porosity

The effect of the selected additives and their respective concentrations on the

porosity is presented in Table 2. Since the porous structure of BC would incorporate the

additive within it, there is a decrease in porosity (p<0.05). There exists a close

relationship between the porosity and the compressive strain range of the samples. The

control (BC) with its higher porosity and large open cellular structure was easier to

deform as compared to other BC composites (Figure 14). A large linear compressive

2 P = Pectin

3 G = Gelatin

4 CSL = Corn steep liquor

5 CS = Corn starch

33

range is due to a high porosity value as more compressive displacement is required for

porous regions to collapse with each other and give a plateau region in the stress-strain

graph. As we see in Figure 12, the linear compressive range decreases with the decrease

in porosity (Table 2).

Table 2: Density and porosity of the selected BC pellicles

Sample Density (kg/m3) Porosity (%)

BC 890 ± 120 41.8 %

BC + CMC 1% 940 ± 220 38.5%

BC + P 1% 1070 ± 190 30%

BC + P 3% 1040 ± 180 32%

BC + G 1% 969 ± 150 36.6%

5.3 BC crystallinity and crystal size

Typical XRD curves are shown in Figure 12. BC shows four main characteristic

diffraction peaks attributed to the crystallographic planes of <100>, <010>, <-1-12> and

<110> at an approx. Bragg angle (degrees) equal to 14.5, 16.6, 20.4, and 22.7,

respectively (Wada & Suciyama, 1993). Cellulose I crystal is almost exclusively found in

native celluloses.

34

Figure 12: Typical XRD curves of BC (Park et al., 2010).

Figure 13 illustrates the fitted X-ray diffraction patterns of BC and BC-

composites together. The control sample looks like typical bacterial cellulose with

distinct <100>, <010>, <-1-12> and <110> peaks. The diffractograms of bacterial

celluloses produced in the presence of the additives are similar to the control cellulose.

The peak width and the intensities of the BC and BC/pectin composite diffractograms are

nearly equal and the diffractogram of BC/pectin composite almost overlaps with the BC,

suggesting no significant interference of pectin in the BC fibril formation (p>0.05). The

difffactograms of celluloses produced in the presence of CMC and gelatin have a

different appearance and lower in intensities as compared to BC. The crystallinity of BC

decreased from 83% to 60% and 71% with the presence of 1% of CMC and 1% gelatin,

respectively. The reduction in the value of crystallinity could be associated with the

creation of amorphous BC or the presence of additional amorphous polymers. The

crystallite size of <110> plane also decreased for every additive. The CMC and gelatin in

the medium may incorporate into BC fibers during fibril formation and crystallization,

35

and thus result in lower crystallinity and crystallite size. Pectin, due to its high molecular

weight, might aggregate more micro-fibrils thus interacting with cellulose at differently

during the assembly process (Gu and Catchmark, 2013).

The crystal size in accordance to width of the <110> crystalline peaks for BC and

BC/composite samples is listed in Table 3. The presence of CMC and gelatin in the

growth medium led to reduction in the lateral dimensions of the cellulose crystals as

evidenced by an increase in the peak widths.

The peak position gives the distance between the unit cell planes (d). The d-

spacing value for our samples based on peak <110> of the fitted crystalline peaks listed

in Table 3. Both CMC and gelatin shifted the position of the <101> and <110> peaks to

higher values of 2θ relative to control cellulose.

The peak intensities of the <010> and <110> peaks in the highly oriented

cellulose differ and are less comparable in contrast to the intensities when

polysaccharides were present in the growth medium. This indicates that the crystallites

BC/additive would be less oriented. Gelatin and CMC form intramolecular hydrogen

bonds when BC microfibrils are polymerizing (Cheng, Catchmark, & Demirci, 2011;

Taokaew et al., 2013). This obstructs the process of crystallization and thereby resulting

in a lower crystallinity. On the other hand, pectin is present in the vicinity of cellulose

microfibrils, without binding chemically to the cellulose. The aggregation step of

microfibrils is not hampered by pectin. The possible reason of pectin not forming bonds

with cellulose is would be the structural difference of the cellulose chain and pectin.

Cellulose is known to have a β-(1, 4)-linkage in the main chain whereas pectin has an α–

linkage (Gu & Catchmark, 2012).

36

Figure 13:X-ray diffraction patterns of BC and BC-composites.

Table 3: Structural features of BC/composites

Sample Crystallinity

(%)

d<110> Crystallite size of

<110> (Å)

BC 84± 3.91 3.93± 0.00 75 ± 1.00

BC+CMC 1% 60 ± 0.61 3.93 ± 0.01 64 ± 4.08

BC+P 1% 82 ± 6.77 3.94 ± 0.01 63.67 ± 4.62

BC+P 3% 83 ± 0.71 3.86 ±0.01 61.33 ± 3.51

BC+G 1% 71 ± 3.09 3.96 ± 0.01 70.67 ± 0.58

0

100

200

300

400

500

600

700

800

900

12 14 16 18 20 22 24 26

Inte

nsi

ty

Two-theta (degrees)

BC

BC-CMC 1%

BC-P1%

BC-P 3%

BC-G 5%

37

5.4 BC Morphology

The supplementation of additives into the media showed morphological changes

on the surface, as shown in Figure 14: FESEM images of BC pellicles at different

magnifications. a), b), c) BC pellicle; d), e), f) BC-CMC 1% Pellicle; g), h), i) BC-Pectin

1%; j), k), l) BC-Pectin 3%; m), n), o) BC-Gelatin 1%. The BC sample is known for its

porous morphology (Cai & Kim, 2010). The images suggests that BC exhibits an

interweaved nanofiber structure and is interconnected to each other and forms a network

structure with the additive as well. On comparing the well-organized structure of BC

microfibrils, the BC-additive microfibrils were less porous. It can be seen that CMC and

gelatin have incorporated in the BC matrix. These additives fill the porous structure of

BC, giving it a more homogeneous texture.

Although the BC fibers are observed on the surface of each composite, the

additives formed a smooth layer over the BC due to their coverage of the BC surface. As

discussed in literature, pectin does not bonds with cellulose and thus aggregates in the

vicinity of BC fibers (Figure 14 (g)–(l)). The pectin might stick on the BC surface

ribbons after the crystallization process. This can be supported by the XRD data (Table

3); pectin does not change the BC crystallinity.

38

Figure 14: FESEM images of BC pellicles at different magnifications. a), b), c) BC pellicle; d), e), f) BC-

CMC 1% Pellicle; g), h), i) BC-Pectin 1%; j), k), l) BC-Pectin 3%; m), n), o) BC-Gelatin 1%

39

5.5 General Discussions

Every polysaccharide has its own way to interact and alter the structure of BC

fibers. Interactions may include hydrogen bonding, electrostatic, ionic, hydrophobic, van

der Waals and even entanglement. From the tensile strength graph (Figure 10), we

observe a knee in the curve of all the composites. The knee, in all sample composites

except BC-pectin 1%, was followed by a drop in stress with increase in strain suggesting

these material began to deform plastically before failure (the samples split in two pieces).

During tensile stretching before the onset of this knee region, the entangled fibers of BC

and pectin were pulled along the extension direction and these randomly arranged fibers

experience alignment parallel to the direction of the applied stress. The aligned fibers can

stand even more load and this alignment increases the tensile strength of the composite.

This pre-alignment imparts elasticity to BC fibers which can be inferred from the long

linear part of the stress strain region (Figure 10). While in case of BC/gelatin composite,

gelatin covers the BC fibers and forms bond with it. Since there is no entanglement, there

is no pre-alignment and thus under pulling force the BC-gelatin composite shows a low

elongation at break. In case of BC-pectin 1% a linear region was observed even after the

knee point. The second linear region had a similar slope as that of the control. This may

occur because in the first linear region the pectin fibers contribute to the tensile modulus.

In the second region many of the pectin-cellulose interactions may break at which point

the BC fibers resist the load more independently and thus the modulus is more

comparable to the control. The BC-pectin 1% composite at break showed no plastic

deformation, i.e., it ruptured without any noticeable prior drop in stress. This property

describes the brittle nature of BC-pectin composite.

40

Water in a BC-additive composite not only binds to cellulose but also binds with

the additive. Water plays a key role as it forms additional bonds with additives. During

compression the water is forced out of the BC sample by the reduction of the volume of

the sample under the applied stress. The resistance of the water leaving the sample is

expected to have a major role in influencing the compression behavior. Although all

materials and samples prepared here are hydrogels, i.e., able to bind and retain water,

especially at high concentrations where gels form, the extent to which each material can

exhibit this behavior varies. The principle issue is the availability of hydrogen bonding

sites per unit mass of the material in the composite. However, the case of composites, the

situation is more complex as additives may both interact with cellulose and water,

forming a more complex system.

Compressive stress-strain curves for BC and BC composites are shown in figure

11. The lowest compression modulus is associated with the pure BC sample. Although

some of the compression behavior may be associated with the stiffness of the cellulose

fiber network, it is hypothesized that the principle factor is the resistance of water leaving

the compressed hydrated sample volume. In this case, the BC sample would exhibit the

lowest compressive modulus since per gram of material it would have the lowest surface

area given that the cellulose fibrils are ordered highly crystalline structures containing

many individual glucan chains. Only the surface groups are available for hydrogen

bonding while glucans in the interior of the fibrils are not. In contrast, the other

biopolymers used to make the BC composites are not ordered or aggregated thus per

gram of material, the additives are able to bind more water than the BC. In general, such

hydrogels made from gelatin, CMC or pectin at concentrations exceeding a few percent is

41

stiff and water cannot be simply squeezed out of them. The water contained in such

hydrogels is completely bound to the biopolymers and under compression such gels will

be destroyed at sufficient stress levels without biopolymer-water separation. Under

compressive stresses, water was forced out of each sample. That water solution was

tested for the presence of any of the biopolymers and in each case no biopolymer was

found in the water removed from the sample. This suggests that in each case, the affinity

of the biopolymer to the BC was of sufficient level to resist the force of the water being

liberated from the polymer. The compressive modulus would then presumably relate

directly the water holding capacity of the biopolymer in the conformational state in which

it exists while bound to the BC. That is difficult to ascertain, but a probable cause could

also be the change in viscosity of the culture medium. The viscosity of culture medium

with no additive (control) is 1.06cP which was increased when 1% CMC, 1% pectin, 3%

pectin and 1% gelatin was supplemented to a value of 5.74 cP, 23.74 cP, 33.28 cP, and

29.41 cP, respectively. The increase velocity might result in a reduced motion of

Acetobacter during BC synthesis and thus alter the BC structure.

42

Chapter 6

General conclusions

6.1 Conclusion

The cultivation of BC in presence of additives changed the morphology and

structure of cellulose. The mechanical properties and the structure the BC so formed were

characterized by tension analysis, compression analysis, FESEM, and XRD.

Incorporation of the additives into the cellulose network resulted in decrease of water

holding capacity of the pellicle. A significant increase in the mechanical properties and a

decrease in the water holding capacity were observed in presence of pectin, gelatin, and

CMC in the media, when compared to the control (BC) (p<0.05). The enhancement of

mechanical properties is because of bonding of cellulose with additive. This study

observed a 20-times significant increase in tensile modulus when 3% pectin was added to

the growth media; tensile modulus increase from 7 kPa to 142 kPa. The compressive

modulus also increase significantly by 3-times after the addition of 1% (w/v) gelatin to

the culture media; increase in value of 7.5 Mpa to 21.94 Mpa (p<0.05). Based on FESEM

images, BC maintains its interweaving property even in the presence of additives.

Cellulose networks formed in BC-additive composites were denser than cellulose

network of BC. Crystallinity of pellicles was reduced in presence of CMC and gelatin

The results showed also showed a noteworthy decrease in crystallinity index

(p<0.05) when CMC and gelatin were added. CMC and gelatin can attach themselves to

nascent cellulose and disrupt the process of crystallization. This disturbs regularity of

micro-fibril structure of BC. Whereas, in case of pectin, it is entangled within the BC

43

fiber mesh but doesn’t interfere with microfibril aggregation. Thus the crystallinity of

BC-pectin is similar to that of control BC.

Since the additives disrupt the orderliness in BC pellicle and alter the

configuration by filling in the voids in its structure, it is likely the additives might make

the BC structure miscellaneous and weak (Taokaew et al., 2013). This could be a possible

reason for the decrease in water holding capacity.

6.2 Suggestions for future work

Polysaccharides are very important material and an extensive research is

required to understand their structural, chemical and mechanical properties. In this

study carboxymethyl cellulose, pectin, gelatin, corn steep liquor and corn starch

were used to study their effects on mechanical properties and morphology of

bacterial cellulose. However, there are varieties of other additives that can be

used. Xanthum gum, chitin, guar gum, and sodium alginate are few other options

for additives. Beyond the additives, this research focused on 3 different

concentrations; 1%, 3% and 5% (w/v) of additive was added to the culture

medium. These concentrations can be tweaked for understanding the effects of

additives on BC properties at different levels of additives. Additionally, more

research could be done to appreciate how much each additive contributes to the

BC structure such that the change in BC properties can be characterized in detail.

Combined effect of two/more additives can also be studied on the

properties of bacterial cellulose. Corn steep liquor (CSL) is a rich nitrogen source

and enhances the BC yield. From our study we found gelatin and pectin to

44

enhance the mechanical properties. One can play with different ratios of either

CSL and gelatin or CSL and pectin in the culture media.

During compression, there is an exchange of mass happening. The water

in the BC-composites was expelled out. Presence of water is an important factor

for compression of these composites and thus the amount of water left during

compression at a particular strain is important. Since water content will be

different at every strain value, experiments can be designed to measure the

amount of water left during compression.

An application of BC-composite could be in biomedical industries.

Increasingly, research in the field of biomedical materials focuses on finding an

effective suture material (Province, 2009). BC can potentially be grown over a

suture to enhance its efficiency in closing and supporting a surgical incision to

support the healing of surgical wounds. BC is biocompatible and has a porous

configuration, which allows the loading of drug molecule over its surface. This

would reduce the risk of surgical site infection and reduce the healing time of

wounds. Moreover, growing of hydrophilic BC over a hydrophobic suture could

be a proposed area for future research. Work can also be done on designing a

polysaccharide blend that will serve as an effective coating on suture material to

promote the growth of BC over its surface.

45

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49

MANMEET SINGH DAYAL

1101 Plaza Drive, State College, PA 16801 +1-814-852-8789 | msd217@psu.edu

EDUCATION

Penn State University Park, State College, PA Graduating: August 2014

M.S. Agricultural and Biological Engineering

G.P.A. – 3.83/4

Jaypee Institute of Information Technology, India August 2008-May 2012

Bachelors of Technology in Biotechnology

G.P.A – 7.2/10

PUBLICATION

Dayal, M.S., N. Goswami, A. Sahai, V. Jain, G. Mathur, A. Mathur. 2013. Effect

of media components on cell growth and bacterial cellulose production from

Acetobacter aceti MTCC 2623. Carbohydrate Polymers 94(1).

CONFERENCE PROCEEDINGS

Mathur, A., G. Mathur, M. S. Dayal, S. Arora, P. Mishra, V. Jain. “Comparative

studies on production and characterization of bacterial cellulose from Acetobacter

sp. and application as carrier for cell culturing”, in Proc. Biomicroworld 2013 (V

International Conference on Environmental, Industrial and Applied Microbiology,

2013, pp. 309