BIODEGRADATION OF NOVEL

CHITIN BIOCOMPOSITES

Suchetana Thakur Chattopadhyay

MASTER OF SCIENCE

SWINBURNE UNIVERSITY OF TECHNOLOGY

2014

i

ABSTRACT

Chitin is a natural polymer found abundantly in structural components of crustaceans

and insects. Commercial interest has been generated in this polymer because of its

widespread availability, high degree of biodegradability; biocompatibility and non-

toxicity.

Chitin and microcrystalline cellulose were combined with different commercially

available polymers to form biocomposites with varying properties. The biodegradation

of these composites was investigated under composting conditions with a view to

develop composites that could be considered as completely biodegradable in accordance

with Australian Standard AS14855 requirements.

A preliminary investigation into biodegradability of chitin/low density polyethylene

(LDPE) composites by the soil burial method indicated faster degradation of the

composites compared to native LDPE. To enhance the biodegradation rate of the

chitin/LDPE composites, an inexpensive, readily-available and partly biodegradable

polymer (Flex 262) was used to partially replace LDPE. Extruded films were tested for

their biodegradability in compost using the respirometric method outlined in AS 14855.

The addition of Flex 262 was shown to increase the rate of biodegradation; however the

degradation was still slow and the extent of degradation limited due to the presence of

non-biodegradable LDPE in the composites.

ii

In order to further explore the potential of chitin fillers, chitin was combined with

polybutylene succinate adipate (PBSA) and polybutylene adipate terephthalate (PBAT)

which are commercially available and fully biodegradable synthetic polyesters. Chitin

and microcrystalline cellulose (MCC) or chitin by itself was combined with the

polyesters in different ratios to form biocomposites. It was found that composites

containing chitin and MCC filler in a PBAT/PBSA matrix degraded at the fastest rate

and was 100% biodegradable (in accordance with Australian Standard AS 14855). To

understand the biological process in greater detail, an assessment of the microbial

community of the resultant composts was undertaken with the help of molecular

biology techniques. In the ‘Day zero’ compost, the dominant microbial community was

found to include members of Phylum Proteobacteria and Day 100 compost was

dominated by members of the group Chloroflexi irrespective of the fact whether

compost contained test composites or not. It can therefore be inferred that the presence

of the composites have not triggered the proliferation of any specialized bacterial

microbiota.

Chitin/PBAT/PBSA composites were also subjected to mechanical testing. It was found

that the tensile strength of chitin filled biocomposites decreased along with increasing

filler content while the reinforcement effect could be observed as an increase in stiffness

(modulus) of the composites with increasing filler loading.

iii

ACKNOWLEDGEMENT

I would personally like to acknowledge the support of the following people for their

contribution to the successful completion of this research:

My supervisors, Dr. Enzo Palombo (Co-ordinating supervisor) and Dr. Mrinal Bhave

for their great and endless support in helping me complete this project.

Dr. Myrna Nisperos (MM Food Company Pty Ltd), Melbourne, who provided both

financial support and assistance in the direction of this project.

Chris Key, Soula Mougos, Ngan Nguyen and James Wang for their assistance in

helping me carry out my laboratory work.

Mike Allan, Dept. of Civil, Environmental & Chemical Engineering, RMIT, for his

assistance with mechanical testing of composites and extending his assistance during a

tough time.

My friends Vandana Gulati, Shanthi Joseph, Saifone Chuaboonmee, Andrew Hunter,

Bita Irani, Elisa Hayhoe, Runyar Memory, Swarnavalli Vaduganathan, Rajeshwari

Vaduganathan, Shakuntala Gondalia, Kaylash Poorun for their constant support while I

was studying at University.

My parents for their inspiration, support and blessings

Finally, a special thank you to my husband Anindya Sankar Chattopadhyay. This thesis

could not have been completed without his constant encouragement.

iv

DECLARATION

To the best of my knowledge, this thesis contains no material that has been accepted for

the award of any other degree or diploma, or written by another person except where

due reference is made in the text of the examinable outcome.

Suchetana Thakur Chattopadhyay

v

TABLE OF CONTENTS

ABSTRACT ............................................................................... I

ACKNOWLEDGEMENT ..................................................... III

DECLARATION .....................................................................IV

TABLE OF CONTENTS ........................................................ V

LIST OF FIGURES ................................................................IX

LIST OF TABLES ..................................................................XI

LIST OF ABBREVIATIONS .............................................. XII

1. INTRODUCTION ................................................................ 1

1.1 Current packaging technology and plastic composites .......................... 1

1.1.1 Polymers sourced from renewable resources........................................ 2

1.1.2 Synthetic polymers with vulnerable groups.......................................... 3

1.2 Biocomposites ............................................................................................. 4

1.2.1 Biodegradation of polyethylene biocomposites .................................... 4

1.2.2 Biodegradation of aliphatic polyester biocomposites ........................... 5

1.3 Chitin .......................................................................................................... 8

1.3.1 Enzymatic degradation of chitin ........................................................... 9

1.3.2 Chitin-based biocomposites ................................................................ 12

1.3.3 Biodegradation of chitin biocomposites ............................................. 13

1.4 Microcrystalline cellulose ....................................................................... 15

1.5 Assessment of biodegradability of plastic packaging ........................... 16

1.6 Polymer-degrading bacterial communities in compost ....................... 18

1.6 Summary .................................................................................................. 19

vi

1.7 Aims of Study ........................................................................................... 19

2. MATERIALS AND METHODS ....................................... 20

2.1 Materials ................................................................................................... 20

2.2 Constitution of chitin biocomposites ...................................................... 22

2.2.1 Chitin/LDPE biocomposites ............................................................... 22

2.2.2 Chitin-Flex262-LDPE biocomposites................................................. 23

2.2.3 Chitin-MCC-PBSA/PBAT biocomposites ......................................... 23

2.2.4 Chitin-PBSA/PBAT biocomposites .................................................... 24

2.3 Masterbatch and blown-film extrusion of chitin biocomposites ......... 24

2.4 Characterization of chitin biocomposites .............................................. 25

2.4.1 Test specimens .................................................................................... 25

2.4.2 Tensile Test ......................................................................................... 25

2.4.3 Notched Izod Impact Test ................................................................... 25

2.4.5 Fourier Transform Electron Microscopy (FTIR) ................................ 26

2.5 Biodegradation of chitin composites ...................................................... 26

2.5.1 Compost .............................................................................................. 26

2.5.2 Bioreactors .......................................................................................... 27

2.5.3 Composting conditions ....................................................................... 27

2.5.4 Organic Carbon analysis ..................................................................... 28

2.6 Molecular techniques for evaluation of microbial populations ........... 29

2.6.1 Buffers and solutions .......................................................................... 29

2.6.2 Isolation of total DNA from compost ................................................. 31

vii

2.6.3 Agarose Gel Electrophoresis .............................................................. 31

2.6.4 Polymerase Chain Reaction ................................................................ 32

2.6.5 Cloning of PCR fragments .................................................................. 32

2.6.6 Sequencing .......................................................................................... 33

2.7 Statistical Analysis ................................................................................... 33

3. MECHANICAL PROPERTIES OF CHITIN

COMPOSITES ........................................................................... 34

3.1 Introduction ............................................................................................. 34

3.2 Results and Discussion ............................................................................ 34

3.2.1 Tensile test .......................................................................................... 34

3.2.2 Impact Strength ................................................................................... 40

3.2.3 Composite morphology....................................................................... 41

4. BIODEGRADATION OF CHITIN COMPOSITES ...... 46

4.1 Introduction ............................................................................................. 46

4.2 Results and Discussion ............................................................................ 47

4.2.1 Biodegradation of chitin/LDPE composites-Test 1 ............................ 47

4.2.2 Biodegradation of chitin-Flex262-LDPE composites-Test 2 ............. 49

4.2.3 Biodegradation of chitin-PBSA/PBAT composites–Test 3 ................ 56

4.2.4 Biodegradation of chitin-MCC-PBSA/PBAT composites–Test 4...... 60

4.2.5 Microbiota associated with biodegradation of chitin-MCC-PBSA/PBAT 64

5. CONCLUSIONS AND FUTURE DIRECTIONS ........... 71

6. REFERENCES .................................................................... 75

viii

7. APPENDICES ..................................................................... 88

ix

LIST OF FIGURES

Figure 1.1 Classification of Biodegradable Polymers....................................................... 2

Figure 1.2 Life cycle of polymers sourced from plants and fermented products ............. 2

Figure 1.3 Structure of PBAT ........................................................................................... 3

Figure 1.4 Structure of PBSA ........................................................................................... 3

Figure 1.5 Biodegradation in composites made with natural fibres and Bionolle 3020 ... 6

Figure 1.6 Structure of chitin and chitosan ....................................................................... 8

Figure 1.7 The figure above is a self-explanatory depiction of the enzymatic network

that exist in microbial cells to degrade chitin into chitooligosachharides and then into N-

acetyl Glucosamine (NAG). OM= Outer membrane, IM= inner membrane .................. 10

Figure 1.8 Electron micrograph image showing chitin digestion by bacteria where the

rounded pits are indicative of enzymatic degradation..................................................... 11

Figure 1.9 The natural breakdown of polymers by microbial depolymerases ................ 16

Figure 2.1 Chitin production flow diagram .................................................................... 20

Figure 2.2 Chitin flakes as received (left) and powdered chitin ..................................... 21

Figure 2.3 Photographs of the respirometric unit showing set-up and direction of airflow

......................................................................................................................................... 28

Figure 3.1 Typical specimens for impact test (top) and tensile test ................................ 35

Figure 3.2 Typical Stress-stain curves for 1803F (PBSA) and its composites ............... 37

Figure 3.3 Typical Stress-stain curves for 2003F (PBAT) and its composites ............... 37

Figure 3.4 SEM characteristics (magnification 200X) of 1803F (PBSA) and its

composites after tensile fracture A) Neat PBSA B) PBSA-CHI/MCC C) PBSAF10 D)

PBSAF20 ........................................................................................................................ 43

x

Figure 3.5 SEM characteristics (magnification 200X) of 2003F (PBAT) and its

composites after tensile fracture A) Neat PBAT B) PBAT-CHI/MCC C) PBATF10 D)

PBATF20 ........................................................................................................................ 45

Figure 4.1 Chitin-Flex262-LDPE composites (test materials for composting study) ..... 50

Figure 4.2 Graph showing percentage mineralization of Chitin- Flex262-LDPE

composites ....................................................................................................................... 51

Figure 4.3 Photographs depicting changes in appearance of films after composting ..... 52

Figure 4.4 Scanning electron microscopy of composite films (300 X magnification) ... 53

Figure 4.5 FTIR Spectroscopy showing changes in EC3 after composting ................... 54

Figure 4.6 Chitin-PBSA composites (test materials for composting study) ................... 57

Figure 4.7 Chitin-PBAT composites (test materials for composting study) ................... 58

Figure 4.8 Graph showing percentage mineralization of Chitin-PBSA/PBAT

composites. ...................................................................................................................... 58

Figure 4.9 Graph showing percentage biodegradation of Chitin-MCC-PBSA/PBAT ... 62

Figure 4.10 Gel showing genomic DNA extracted from compost at Day 0 (Lane 1) and

DNA ladder (Lane 2; Hyperladder I) .............................................................................. 66

Figure 4.11 Gel showing PCR product of the expected size (~ 900 bp) for Day 0

compost in Lane 2. Lane 1 contains DNA Hyperladder I ............................................... 66

xi

LIST OF TABLES

Table 1.1 Weight loss in PE-chitin composites .............................................................. 14

Table 2.1 Physical properties of chitin ............................................................................ 21

Table 2.2 Composition of Biopla plastic films ............................................................... 22

Table 2.3 Composition of EC films ................................................................................ 23

Table 2.4 Composition of chitin-MCC-PBSA/PBAT composites .................................. 23

Table 2.5 Composition of chitin-PBSA/PBAT composites ............................................ 24

Table 2.6 Weight of Sample, MCC and Compost for different composting tests .......... 27

Table 2.7 Buffers and solutions used for nucleic acid extraction and agarose gel

electrophoresis................................................................................................................. 29

Table 2.8 Buffers and solutions used for DNA sequencing ............................................ 29

Table 2.9 Solutions and media for cultivation of bacteria and fungi .............................. 30

Table 3.1 Tensile strength and modulus of chitin filled PBSA and PBAT composites at

different filler loadings .................................................................................................... 36

Table 4.1 Validation criteria for AS14855 ...................................................................... 49

Table 4.2 Percentage biodegradation of Chitin-Flex262-LDPE composites. ................. 51

Table 4.3 Validation criteria for AS14855 ...................................................................... 57

Table 4.4 Percentage mineralization of Chitin-PBSA/PBAT composites. ..................... 59

Table 4.5 Validation criteria for AS14855 ...................................................................... 61

Table 4.6 Percentage mineralization of Chitin-PBSA/PBAT composites ...................... 62

Table 4.7 Microbial diversity in Day 0, Day 100 Blank and Day 100 PBAT composts 68

xii

LIST OF ABBREVIATIONS

AGRF Australian Genome Research Facility

Ltd

AS Australian Standards

ASTM

American Standard Testing Method

CFU Colony Forming Units

DNA Deoxyribonucleic Acid

DSC Differential Scanning Calorimetry

EDTA Ethylenediaminetetraacetic Acid

FTIR Fourier Transform Infrared

IPTG Isopropyl -D-1 thiogalactopyranoside

ISO International Organization For

Standardization

LB Luria Bertani

LDPE Low Density Polyethylene

MCC Microcrystalline Cellulose

MSW Municipal Solid Waste

MW Molecular Weight

PBAT Poly (Butylene Adipate-Co-

Terephthalate)

PBS Polybutylene Succinate

PBSA Poly (Butylene Succinate-Co-Adipate)

PCL Polycaprolactone

PCL Polycaprolactone

PCR Polymerase Chain Reaction

PE Polyethylene

PHA Poly(Hydroxyl Alkanoate)

PHB Poly(Hydroxyl Butyrate)

PHBV Poly(Hydroxyl Butyrate-Valerate)

xiii

PLA Poly(Lactic Acid)

PVA Poly(Vinyl Alcohol)

RHF Rice Husk Flour

RNA Ribonucleic Acid

SDS Sodium Dodecyl Sulphate

SEM Scanning Electron Microscopy

SOC Super Optimal Broth

TAE tris-Acetate EDTA

tris-HCL tris-Hydrochloride

UV Ultraviolet

WF Wood Flour

X-GAL 5-Bromo-4-Chloro-3-Indolyl-Βeta-D-

Galactopyranoside

1

1. INTRODUCTION

1.1 Current packaging technology and plastic composites

Packaging waste can be subdivided into synthetic and paper packaging (Jayasekara et

al. 2005). Non-biodegradable synthetic packaging is designed for long-term use; hence,

its application in disposable materials is seen as a wasteful process. Additionally, mass

recycling of synthetic packaging, which is often contaminated with food and other

substances, involves huge cost and is practically impossible which leads to the majority

of packaging waste ending up in landfill (Jayasekara et al. 2005). Biodegradable

packaging (BP) partly resolves the issues surrounding the huge amount of plastics going

to landfill since these materials require much less expenditure for ecological disposal

(Dřímal et al. 2007). Over the past three decades, scientists have investigated the

development of plastics that will degrade in a predictable manner due to environmental

stresses (Wollerdorfer and Bader 1998; Mohanty et al. 2000). As restrictions regarding

the use of plastic products increase (Zerowaste 2009; Martin 2012), the demand for

production of environmentally friendly packaging will also increase. However,

production of BPs must be accompanied by competitive pricing and infrastructure

development for their suitable disposal, such as efficient composting and other

bioconversion facilities. The lack of such infrastructure will most likely lead to BPs

being dumped in a sterile and dry landfill environment.

BP has been mainly developed for three major areas: agriculture, surgery and consumer

packaging (Gross and Kalra 2002). Recently developed ‘biodegradable’ packaging falls

into four main groups: synthetic polymers with additives, synthetic polymers with

vulnerable groups, polymers sourced from natural sources and composites which are

made by blending materials obtained from two or more of the above-mentioned sources

(Mohanty et al. 2000) (Figure 1.1).

2

‘This image is unable to be reproduced online. Please consult print copy held in the

Swinburne Library’

Figure 1.1 Classification of Biodegradable Polymers

(Adapted from Weber 2000)

1.1.1 Polymers sourced from renewable resources

Biopolymers from renewable sources have attracted much attention in recent years as

they provide a sustainable and ecologically attractive alternative to packaging products

sourced from non-renewable resources. Among the multitude of renewable resources

that have been investigated for application in packaging technology , the major ones are

starch, polylactic acid (PLA), Polyhydroxy alkanoates (PHA), xanthan, pullulan,

cellulose, flax, hemp, jute and ramie (Mohanty et al. 2000; Gross and Kalra 2002;

Averous and Boquillon 2004). Fig. 1.3 depicts the life-cycle of a representative group of

renewable biopolymers. Starch-based BPs are promising because of the low cost and

widespread availability of starch. However, their sustainability is questioned since they

are essentially made from food crops (corn) which not only occupy extensive farmland

can be of the genetically modified crop (GMC) variety. Without proper impositions,

such modified ‘plastic crops’ can mingle with the food supply raising serious

environmental and health issues (Moschini 2006). ‘Plastic crops’ also require the use of

huge amounts of pesticide and fertilizers and have to compete with biofuel crops for

space (Thomas 2008).

Over the last few years, cellulosic and lignocellulosic waste products derived from plant

and animal sources have attracted much attention from researchers for their applicability

in the biodegradable polymer industry and for furthering the concept of waste to

resource conversion (Figueiredo et al. 2005; Shih et al. 2006; John and Thomas 2008;

Wu 2012)

‘This image is unable to be reproduced online. Please consult print

copy held in the Swinburne Library’

Figure 1.2 Life cycle of polymers sourced from plants and fermented products

(Adapted from Gross and Kalra 2002)

3

1.1.2 Synthetic polymers with vulnerable groups

Several aliphatic polyesters such as poly- (ε-caprolactone) (PCL), polylactide,

polyglycolide and copolymers are used as BPs because of their biodegradability and

hydrophobicity (Hoshino and Isono 2002; Kuan et al. 2006; Somiya and Sakai 2006).

Poly (butylene succinate-co-adipate) (PBSA) and poly (butylene adipate-co-

terephthalate) (PBAT) are aliphatic polyesters which are produced from petroleum-

based resources and are biodegradable according to ASTM D5338 (2003) specifications

(Kijchavengkul et al.; Fujimaki 1998). PBSA is produced by melt condensation reaction

of glycols such as 1, 4- butanediol and acids such as succinic acid and adipic acid.

PBAT is a linear random copolyester of 1,4- butanediol and terepthalic acid (BT) and

1,4- butandiol and adipic acid (BA) (Kijchavengkul et al.2008).

‘This image is unable to be reproduced online. Please consult print copy held in the

Swinburne Library’

Figure 1.3 Structure of PBAT

(Adapted from Shi et al. 2005)

‘This image is unable to be reproduced online. Please consult print copy held in the

Swinburne Library’

Figure 1.4 Structure of PBSA

(Adapted from Baidurah et al. 2005)

Because of the excellent material properties they exhibit, PBSA and PBAT have been

involved in commercially successful applications (Chen and Zhang 2010; Olivato et al.

2012). Unfortunately, widespread commercialization of these polymers is limited

because they are relatively expensive compared to non-biodegradable petroleum based

polymers (Wu 2012).

4

1.2 Biocomposites

A composite is a multi-phase material made up of two or more physically distinct

components. One is the matrix phase which is a continuous phase and the other is a

dispersed phase. The disperse phase is made up of the ‘filler’ material. In the case of

fibre-reinforced composites, glass fibres have been used traditionally. Fibre-reinforced

composites are widely used to build various complex structures such as tubes, car door

interior panelling and electronic components (Mohanty 2000).

Natural materials such as plant-based cellulosic fibres like jute (Wollerdorfer and Bader

1998; Mohanty et al. 2000; Liu et al. 2009), flax (Van de Velde and Kiekens 2002),

abaca (Shibata et al. 2002), hemp, sisal , kenaf, coir and waste materials such as

newspaper, rice husk and sugarcane bagasse (Averous and Boquillon 2004) have been

incorporated into polymer composites as filler materials and as replacement for glass

fibres in various studies because of their inherent capacity to degrade in natural systems.

Blending is an especially important process for developing industrial applications of

composite polymeric materials and compatibility among components has a marked

influence on the physical properties of polymer blends. The physico-chemical properties

of composites have been found to be more than just a superposition of the components

(Mohanty et al. 2000; John and Thomas 2008). Natural fibres have low density and

stiffness and allow the production of low density composites with higher filler

concentration (Spoljaric et al. 2009).

1.2.1 Biodegradation of polyethylene biocomposites

This group contains composites made of polyethylene (high molecular weight and non-

biodegradable) combined with additives that are readily consumed by microorganisms.

Various attempts have resulted in the production of ‘biodegradable’ packaging made of

mainly polyethylene (PE) with starch (Evangelista et al. 1991; Hakkarainen et al. 1997;

Wang et al. 2004). There exists a significant volume of literature which indicates that

these materials are not fully biodegradable since the remaining synthetic component

(PE) undergoes very little or no microbial attack (Evangelista et al. 1991; Chandra and

Rustgi 1997; Ratajska and Boryniec 1999). However, other investigations have pointed

out that degradation of filler materials lead to weakening of the matrix (PE) which

5

becomes susceptible to further deterioration (Ołdak et al. 2005). Blends of cellulose and

polyethylene were prepared with varying concentrations of cellulose ranging from 5%-

30% and their photo and biodegradation properties were found to be vary according to

the amount of filler. The difference in behaviour has been attributed to the varying

interaction between the macro chains of both the components, due to a difference in the

dispersion of cellulose particles in the PE matrix (Ołdak et al. 2005).

1.2.2 Biodegradation of aliphatic polyester biocomposites

Aliphatic polyesters can be quickly degraded by fungi and bacteria in present in natural

environments (Ikada 1999; Maeda et al. 2005). They are commercially produced on a

large scale but their application is hindered due their high pricing. The presence of

cheap ‘fillers’ can help to alleviate the high cost as well as impart special characteristics

to the composite thus formed. Kim et al. (2006) developed composites with rice husk

flour (RHF) and polybutylene succinate (PBS) and subjected them to biodegradation in

two different environments, simulated municipal solid waste (MSW) and soil. In both

environments, it was found that the percentage weight loss in RHF composites was

higher than the neat PBS which the authors attributed to superior mineralisation of

lignin and cellulose in RHF. However, the weight loss was around 18% in MSW

compared to 7% in soil in a period of 80 days. Microbial plate counts showed almost

twice the number of colony forming units (CFU) in MSW and this could be one of the

main reasons for higher weight loss of composites in MSW.

Abaca fibre (5 mm) and surface treated (acetylated) abaca fibre (AA-abaca) was melt

blended with different aliphatic polyesters PCL, PBS, PLA and PHBV. The addition of

abaca fibres caused PBS and PHBV composites to degrade faster than neat PBS and

PHBV. However, the weight loss was limited in case of composites having AA-abaca.

The authors attributed the higher weight loss to the presence of cracks which had

developed on the surface of abaca-polyester composites that were absent in AA-abaca-

polyester composites. Hence, surface irregularities which occurred due to mixing of two

immiscible components enhanced biodegradability. The study hinted that AA-abaca-

polyester composites were stronger in terms of tensile strength than abaca-polyester

composites. Surface treatment, in this case acetylation of the fibres, made them less

polar and increased the hydrophobicity of the fibres by acetylating the hydroxyl groups

in the cellulosic components. This made the fibres much more compatible and there was

6

a better dispersion of the abaca fibres in the matrix. However, this also made the

composite less susceptible to biodegradation in soil.

Evangelista et al. (1991) proposed that modification of starch increased the miscibility

of starch/LDPE films but reduced the biodegradation dramatically compared to

unmodified starch/LDPE blends. It was also found that miscibility of PCL in LDPE

matrix was inversely proportional to its biodegradation by Rhizopus arrhizus lipase.

However, studies of some other polymer composites indicate quite the contrary. Starch-

based polyester biocomposites have found their way into numerous commercial

products which are marketed by companies like Mater-BiTM and Plantic

TechnologiesTM. It has been found that addition of starch to neat resins increases the

rate of mineralisation of composites (Halley et al. 2001; Jayasekara et al. 2003;

Mohanty and Nayak 2009). PLA/starch blends which were compatibilized with maleic

anhydride (leading to superior adhesion between the two) showed higher

biodegradability than ordinary PLA/ starch blends having the same PLA content (Jang

et al. 2007). It was observed that this happened even though the crystallinity of the

maleic anhydride compatibilized blend was much higher. The authors proposed that the

reaction between freely available water in the compost and maleic anhydride resulted in

the formation of acids which increased the chain scission of PLA by hydrolysis

resulting in a higher rate of biodegradability.

‘This image is unable to be reproduced online. Please consult print copy held in the

Swinburne Library’

Figure 1.5 Biodegradation in composites made with natural fibres and Bionolle 3020

(Adapted from Tserki et al. 2006)

7

A study evaluating starch-blended PBSA 3001 found that incorporation of starch

significantly increased the rate of biodegradation of these composites (Ratto et al.

1999). Pure PBSA3001 had a half-life of 231 days in compost and incorporation of 5%

starch dramatically reduced it to 80 days. The rapid decrease in molecular weight during

composting was attributed to disruption of ester linkages by starch particles which

might have made the composite films much more susceptible to hydrolytic enzymes. In

another study, small amounts of recycled wood fibres and nanoclays were added to

PHBV/PBAT blend to enhance the material properties of brittle PHBV/PBAT (Javadi et

al. 2010) and to study the effect of fibre content on the biodegradability of composites.

The order of higher weight loss was found to be PBS/10% jute composite > PBS/20%

jute composite > PBS/30% jute composite > pure PBS film > bulk jute fiber. However,

similar to PLA composites, the tensile properties of PBS and its derivatives are

compromised due to addition of fibres.

Kim et al. (2006) prepared RHF and wood flour (WF) filled PBS biocomposites and

reported that the addition of agro-flour to PBS produced a more rapid decrease in the

tensile strength and notched Izod impact strength, contrasting with rapid increase in the

percentage weight loss of the biocomposites during the natural soil burial test.

Similar trends were observed by other researchers (Mohanty et al. 2000; Liu et al. 2009;

Wu 2012) who found that chemical modification of natural fibres increased the

compatibility between hydrophilic fibres and the hydrophobic matrix leading to

improved tensile properties. Therefore, it can be concluded that natural fillers, such as

cellulose and starch, form a network which allows the movement of water into the

biodegradable polyester matrix leading to its higher rate of degradation (Fig. 1.7).

Blending and compatibilization is very important particularly in the development of

new biodegradable materials. Among polymer scientists, it is generally accepted that

there is a conflict between tensile properties and biodegradability in natural fibre based

composites and the balance between the two should be taken into consideration while

deciding the application of such composites.

8

1.3 Chitin

Since chitin is the focus of the work described in this thesis, a detailed review of its

properties is presented. Chitin is a natural polymer found abundantly in structural

components of crustaceans and insects and cell walls of many species of fungi. It is the

second most abundant natural polymer on the earth preceded only by cellulose. It is an

insoluble homopolymer of β (1-4)-linked N-acetyl-D-glucosamine (IUPAC: 2-

acetamido-2-deoxy-D-glucose) and is produced in enormous quantities particularly in

the exoskeleton of crustaceans, molluscs, arthropods and cell walls of fungi (Gooday

1990). A modification of chitin is its deacetylated form, chitosan, which is a polymer of

β (1-4)-linked D-glucosamine (IUPAC: 2-amino-2-deoxy-D-glucose). Chitin has a

degree of N-acetylation (DA) > 50% and is insoluble in water (Kasaai 2009). According

to X-Ray diffraction studies, chitin exists in three polymorphic forms α, β and γ

differentiated with respect to arrangement of chains either parallel, antiparallel or

mixture of both in the crystal form (Dahiya et al. 2006).

‘This image is unable to be reproduced online. Please consult print

copy held in the Swinburne Library’

Figure 1.6 Structure of chitin and chitosan

(Adapted from Jayakumar et al. 2011)

Chitin is a waste product of the seafood processing industry. Although a renewable

resource of value, it is discarded into the landfills or dumped into the oceans due to

apparent lack of utility. It can also be harvested from cell walls of fungi where its

quantity and generation is supposedly higher than animal chitin (Ugrozov et al. 2008). It

is reported to be produced commercially in India, Japan, Poland, Norway and Australia.

It is a hard, inelastic polysaccharide resembling cellulose except for the higher

percentage of nitrogen (6.89%). Commercial interest has been generated in this polymer

because of its high degree of biodegradability, biocompatibility and non-toxicity (Ravi

Kumar 2000). Chitin and chitosan does not occur in humans which renders them slowly

degradable and non-bioabsorbable in the gastrointestinal tract (lack of chitin degrading

9

enzymes). Hence chitin metabolism is regarded as an important target area for

development of drugs and pesticides (Horn et al. 2006).

However, chitin is difficult to process because of its extreme insolubility. A variety of

solvent systems have been used for spinning chitin fibres, including 5% Lithium

chloride (Ravi Kumar 2000). Most chitin film preparations rely on solution casting

method whereby the chitin-containing solution is made solvent-free and residual chitin

is subsequently dried to give chitin films (Yusof et al. 2004). Gels, films, fibers and

sponges are made from chitin or its derivatives (to overcome the rigidity of chitin) and

often involve the addition of plasticizers and other chemicals.

Chitin derivatives and chitosan are being used in a number of industries (Kandra et al.

2012) and are especially useful as chelating and flocculating agents for wastewater

treatment, animal feed additives, wound dressing, cosmetics , fungicides, anti-microbial

food packaging (Ouattara et al. 2000; Coward-Kelly et al. 2006) and as biosorbents

(Ugrozov et al. 2008).

1.3.1 Enzymatic degradation of chitin

Degradation of chitin is brought about by chitinases. They were first identified in 1911

as an antifungal factor in orchid pulp followed by a similar factor being isolated from

snails in 1929 by Karrer and Hoffman (Felse and Panda 1999). Chitinases are divided

into two categories based on mode of action: endochitinases and exochitinases. These

two groups of enzymes generate a pool of chitobiose and N-acetylglucosamine which

the cells uptake and metabolize or use for the biogenesis of cell walls (Howard et al.

2003).

10

Endochitinases [EC 3.2.1.14] cleave the glycosidic linkages randomly at internal sites

giving rise to many low molecular weight, soluble dimers, trimers or tetramers, like di-

acetyl chitobiose, chitotriose and chitotetraose. Exochitinases cleave the glycosidic

linkages from the non-reducing end of the chitin fibril generating di-acetyl chitobiose

units (Cohen-Kupiec and Chet 1998). These enzymes have been isolated from a large

number of viruses, bacteria, fungi, insects, plants and animals (Dahiya et al. 2006) and

they have been grouped into two families of glycosyl hydrolases, family 18 and family

19, based on the amino acid sequence similarities of their catalytic domain (Cohen-

Kupiec and Chet 1998).

In vertebrates, chitinases help in digestion of fungi and invertebrates which can form

part of their diet. These enzymes are present in the animal itself or secreted by gut

microbiota, namely Vibrio and Photobacterium and enterobacteria in fish, while in

insects and crustaceans, chitinases help in morphogenesis by shedding of the old

exoskeleton/cuticle (Gooday 1990). In fungi, chitinases are implicated in

morphogenesis as well as nutrition. Plants produce chitinases mainly as antifungal

weaponry but, in case of winter rye, it is a proven anti-freeze protein (AFP) as well

(Yeh et al. 2000) .

‘This image is unable to be reproduced online. Please consult print copy held in the

Swinburne Library’

Figure 1.7 The figure above is a self-explanatory depiction of the enzymatic network that exist in microbial cells to degrade chitin into chitooligosachharides and then into N-acetyl Glucosamine (NAG). OM= Outer membrane, IM= inner membrane

(Adapted from Howard et al. 2003)

Chitinases are ubiquitous in bacteria with a number of authors reporting the isolation

and purification of the same (Fig. 1.10). These bacteria are present in the marine,

freshwater and terrestrial ecosystems. Marine ecosystems produce the highest amount of

chitin therefore it is not surprising that a large number of bacteria have been found in

the oceans which can degrade chitin. According to one estimate, around 10% of marine

bacterial populations could be supported by chitin which acts as a source of energy

(Kirchman and White 1999). Chitinases in general have attracted a lot of attention

mainly because of their antifungal and insecticidal properties since chitin is a major cell

11

wall component in fungi and present in the exoskeleton of insects. A number of reviews

exist on the mycolytic actions of chitinases isolated from various bacteria and fungi

(e.g. the fungus Trichoderma harzianum) (Gokul et al. 2000; Patil et al. 2000).

‘This image is unable to be reproduced online. Please consult print copy held in the

Swinburne Library’

Figure 1.8 Electron micrograph image showing chitin digestion by bacteria where the rounded pits are indicative of enzymatic degradation

(Adapted from Keyhani and Roseman 1999)

12

1.3.2 Chitin-based biocomposites

Although full of potential, chitin is difficult to process, due to its high crystallinity,

thermal degradation before melting and a lack of solubility. To explore their full

potential, special emphasis has been put on chemical modification of chitin and chitosan

by acetylation, etherification and graft copolymerization (Honma et al. 2006).

The alternative way to utilize chitin is to combine it with other polymers. Although the

surge in chitin production is relatively recent, researchers have already looked into

various aspects of utilizing chitin and its derivatives as filler material in biocomposites

due to its highly sought after properties (section 1.3). Rizvi et al (2011) used chitin

powder from crab shells to prepare PLA-chitin composites which showed that

incorporation of 5 % (w/w) chitin led to an increase in Young’s modulus and a decrease

in Ultimate Tensile Strength (UTS) and strain-to-failure. The reason was cited as

hydrolysis of PLA due to absorbed moisture that occurred during melt-processing of

PLA and chitin. In fact, PLA has the disadvantageous attributes of low melt strength

and susceptibility to hydrolysis during processing (Petinakis et al. 2010). In another

study, polycaprolactone and chitin fibres (1cm long) were melt blended and

compression molded. It was observed that increasing the amount of chitin content led to

significant increases in Young’s modulus, storage modulus and tensile strength (Chen et

al. 2005). Chitin has also been blended with PVA and PBSA to make composites for

biomedical applications due to its regenerative properties (Cho et al. 2001)

13

1.3.3 Biodegradation of chitin biocomposites

Very few studies have been conducted on biodegradation of chitin biocomposites.

Takasu et al. (1999) blended Chitin-graft- poly(2 methyl-2-oxazoline) and Chitin-graft-

poly(2-ethyl-2-oxazoline) with PVA and obtained a film about 40 microns thick by

drying the solution on a Teflon laboratory dish at 50°C and then in vacuum at the same

temperature. The degradation was tested by burying films (20mm×10mm×0.04mm) in

soil which had been composted for more than 10 years. The soil (pH 7.1-7.4) was kept

in a controlled atmosphere with 70-80% relative humidity and 27°C temperature for

several months. After recovery, the polymers were dried to constant weight under

reduced pressure and the weights were determined. Biodegradation was found to be 11-

47% after 150 days. Graft copolymers of chitosan and polymethylmethacrylate (C-g-

PMMA) were prepared and heat pressed between Teflon sheets to form thin films

(Harish Prashanth et al. 2005). Biodegradation was studied by inoculating C-g-PMMA

powder in Czapek’s solution with humus soil with the co-polymer at 2.0 g/L

concentration. After 10 days, Aspergillus flavus was isolated from the broth and co-

polymer pieces (3cm×3cm, 270% grafting) were inoculated with fungal spores and

incubated at room temperature. The percentage degradation of the powder was 45% in

25 days.

In another study, (Makarios-Laham and Lee 1995) the biodegradation of

polyethylene/chitin (PE-chitin) and polyethylene/chitosan films was studied by using

them as the sole carbon and nitrogen sources for pure cultures of Serratia marcenscens,

Pseudomonas aeruginosa and Beauveria bassiana. Films were made by combining low

density (0.918) granulated PE with 10% practical grade chitin/chitosan powder (particle

size 500 microns) and heat sealing the mixture between hot plates with the resulting

film having thickness of 0.25-0.35mm. Strips of either film were placed in seeded Petri

plates and inoculated with each culture. The percentage weight loss was assessed after

two, four, six and eight weeks of inoculation. To assess the effect of natural soil

organisms, film samples were placed in agricultural soil contained in pots with one set

being kept in the laboratory and the other set in open field conditions. Films were

recovered for weight loss assessment after three and six month intervals. Table 1 shows

the degradation observed in PE-chitin films.

14

Table 1.1 Weight loss in PE-chitin composites

(Adapted from Makarios-Laham and Lee 1995)

Inoculated with/buried in Percentage weight loss

Aspergillus flavus spores 45% in 25 days.

Soil composted for more than 10 years 11-47% in 150 days

Soil composted for more than 10 years 16-49% in 150 days

Beauveria bassiana culture 11.22% in 60 days

Serratia marcescens culture 10.92% in 60 days

Pseudomonas aeruginosa culture 9.43% in 60 days

Soil under laboratory conditions 8.47% in 180 days

Soil under open field conditions 10.45% in 180 days

15

1.4 Microcrystalline cellulose

Microcrystalline cellulose powder (MCC) is produced by spray drying of hydrolysed

wood pulp to produce particles with average size 20-90µm. It has been used mainly in

the food industry as a texturiser, emulsifier, extender, anti-caking agent and bulking

agent (FAO, 1997). Cellulose is a molecule with both crystalline and amorphous

regions. The crystallinity index is used to measure the proportion of crystalline regions

in cellulose and MCC has high crystallinity index ranging from 70% to 80% (Bansal et

al. 2010). Microbial degradation of MCC occurs extracellularly and is catalyzed by

secreted cellulases. These secreted cellulases include mainly endo-1,4-β-glucanase, exo-

1,4-β-d-glucanase and β-1,4-glucosidase, where endo-1,4-β-glucanase cleaves the linear

β-1,4 glucose bond first, exo-1,4-β-d-glucanase cleaves a cellobiose unit from the end of

the polymer and finally β-1,4-glucosidase hydrolyses cellobiose and short-chain β-1,4-

glucose polymers into glucose (Kato et al. 2004).

Microcrystalline cellulose has been shown to act as an excellent filler material for

constructing polymer composites with desired chemical and physical properties (John

and Thomas 2008) and to make the composite more ecofriendly. Microcrystalline

cellulose (MCC) 1-10% has been used as reinforcement material in a polypropylene

matrix. The compatibility between the two phases was improved by the addition of poly

(propylene-graft-maleic anhydride) and surface treatment of MCC to make it more

hydrophobic. Enhancement of thermal stability (until 4%) and crystallisation

temperature (Tc) was observed. Composites with 30% cellulose were found to be more

susceptible to photo and biodegradation (Ołdak et al. 2005). Maiti et al. (2011) used

10% MCC to reinforce PVA-starch-glycerol composites and found that there was a

significant increase in mechanical strength when compared to unreinforced composites.

MCC reinforcement improved tensile strength, storage modulus and glass transition

temperature of MCC-thermoplastic starch composites (Ma et al. 2008). This could be

attributed to large surface area of microcrystalline cellulose particles and polar groups

on cellulose molecules (-OH) which allows for hydrogen bonding between the fillers

and the matrix (Ramires et al. 2010).

16

1.5 Assessment of biodegradability of plastic packaging

Biodegradable packaging partly resolve the issues surrounding the huge amount of

plastics going to landfill since on after use these materials require little financial

expenditure for ecological disposal and pose no harm to the environment and wildlife

(Dřímal et al. 2007). However, protocols are required to correctly assess their

degradation in the environment. Two biodegradation conditions, aerobic and anaerobic,

are categorized by the presence or absence of oxygen (O2), respectively. Biodegradation

in a compost pile is predominantly aerobic. In contrast, anaerobic biodegradation

happens in an oxygen-absent environment. Instead of CO2, methane gas (CH4) and

water are generated and released. Examples of anaerobic conditions include those in

sewage and in landfills where methane is collected.

Laboratory or field studies to understand biodegradation of plastics have been

performed since plastics were first invented. There are a range of methods currently

available to test biodegradation based on direct measurements like weight loss and

FTIR/DSC/SEM/contact angle measurement analysis of degraded samples or indirect

measurements such as consumption of O2/amount of CO2 produced (Jayasekara et al.

2005). Most of the methods available for material degradation are based on

determination of CO2, a by-product of microbial assimilation of carbon present in the

plastics (Calmon et al. 2000). The ASTM D 5338 (2003) and the AS ISO 14855-2005

(2005) are the two most popular examples of standard methods that help to determine

the aerobic biodegradability of plastic materials in compost. The method simulates

typical aerobic composting conditions in a municipal organic waste composting facility

where conditions like temperature, moisture and aeration are strictly controlled.

‘This image is unable to be reproduced online. Please consult print

copy held in the Swinburne Library’

Figure 1.9 The natural breakdown of polymers by microbial depolymerases

(Adapted from Gu 2003).

17

As shown in Fig. 1.11, the environmental conditions dictate the degradative pathway.

The presence of O2 leads to the formation of CO2, H2O and biomass whereas inorganic

conditions lead to formation of CH4 and CO2 as well. Aerobic processes leads to higher

rate of degradation because it is known that O2 is a thermodynamically better electron

acceptor than SO42− or CO2 because of which the process yields much more energy and

supports an abundantly diverse microbiota (Gu 2003).

According to the above standards, it is assumed that some of the carbon present in the

plastics is used by micro-organisms as a source of carbon/food and that, after

respiration, the carbon is mainly released as CO2. Depending on the quantity of the

carbon in the plastics that is converted to CO2 by this natural process, the plastic is

judged to be biodegradable or otherwise. Cellulose is used as a reference material since

it is consistently degraded by over 70% in 45 days when standard protocols are

maintained (Jayasekara et al. 2005).

18

1.6 Polymer-degrading bacterial communities in compost

Biodegradable polymers such as PBSA and PBAT are hydro-biodegradable which

means that microbial assimilation sets in after hydrolytic breakdown of large polymer

chains into smaller chains. A few studies have been carried out on PBSA and PLA-

degrading bacteria (Hayase et al. 2004 and Ishii et al. 2008) and some strains have been

isolated (Kim et al 2007; Teeraphatpornchai et al. 2003), majority from soil. Although

composting is one of the favoured methods for degradation of polymers and

biocomposites, the microbial community involved in biodegradation of polymers in

compost has not been studied as extensively as in soils (Sangwan and Wu, 2008). Along

with high temperature and moisture content, composts support diverse microbial

communities which are implicated in the rapid breakdown of biodegradable materials.

Thermophilic bacteria in composts are capable of assimilating low molecular weight

products that result from hydrolytic dissolution of the materials and their identification

can lead to new frontiers in designing 100% biodegradable polymers and/or discovery

of superior environments for plastic waste disposal (Sangwan and Wu, 2008).

Polymer degradation using cultivation-dependent methods, such as most probable

number (MPN), plate counts, selective enrichment, or clear-zone formation have been

investigated (Pranamuda et al. 1997; Pranamuda and Tokiwa 1999) However, only a

small fraction of the microorganisms present in environment samples can be cultured

using these classical cultivation methods because they are biased towards cultivation of

specific groups of fast-growing microorganisms or those adapted to grow under

particular laboratory conditions (Peters et al. 2000, Quaiser et al. 2003). Molecular

approaches to the identification of bacteria show promising results. The amplification of

16S rDNA of any bacterial species is possible without prior cultivation when broad-

range PCR primers targeted to highly conserved regions are applied thus circumventing

some of the limitations of the cultivation approach (Peters et al. 2000). The comparison

of amplified and sequenced 16S rDNA sequences with sequences of known bacteria in

16S rDNA databases facilitates a subsequent phylogenetic identification (Schabereiter-

Gurtner et al. 2001).

19

1.6 Summary

It is clear from the literature that a broad range of polymers and composites have been

developed to overcome the waste disposal issues associated with the use of ‘non-

biodegradable’ synthetic plastic materials. Chitin, on being a cheap and versatile waste

product, composites containing varying amounts of chitin and its derivatives have been

developed for wide ranging applications that include wastewater treatment, cosmetics,

wound healing and food packaging. However the effects of chitin and other cellulosic

fibres in combination with synthetic biopolymers on biodegradability and mechanical

properties are yet to be studied in detail. On one hand, the biodegradability of chitin is

well documented yet on the other hand little is known about the environmental

biodegradation of chitin biocomposites. Moreover the effect of biodegradation of chitin

composites on natural microbiota under composting conditions has not been

investigated. A detailed study of the structure and biodegradation of these novel

composites is therefore imperative in order to develop and design commodious

materials from chitin.

1.7 Aims of Study

The aims of the study are the following:

1. Developing novel chitin containing biocomposites that are potentially

biodegradable and compostable.

2. To investigate mechanical properties of the composites e.g. tensile strength and

increase in tensile modulus.

3. To determine the biodegradability of the biocomposites utilizing the principles

of AS ISO 14855 (2005) which measures carbon-di-oxide evolved during the

breakdown of biocomposites under controlled composting conditions.

4. To identify and track changes in bacterial population and diversity before and

after composting, in order to evaluate any effect of the biodegradation on the

microbiota of the compost, using molecular ecological techniques and to identify

any particular bacterial species of interest for further applications.

20

2. MATERIALS AND METHODS

2.1 Materials

Chitin flakes were purchased from N.G. Alexander and Co. Pty Ltd., Armadale,

Victoria, Australia. The method of manufacturing is outlined in Fig. 2.1.

Figure 2.1 Chitin production flow diagram

(Adapted from material testing document provided by N.G. Alexander and Co. Pty Ltd.,

Victoria, Australia 2009)

Shell of crab or shrimp

Calcium removed by HCl

treatment

Protein removed by NaOH

treatment

Chitin

Drying

Identification

Packaging

Wash with water to pH 7

Wash with water to pH 7

21

The physical properties of the chitin flakes are outlined in Table 2. Chitin flakes were

ground by a mechanical grinder and sieved to 300 mesh sizes. This powdered form of

chitin (Fig. 2.2) was used to manufacture all the biocomposites used in this study.

Figure 2.2 Chitin flakes as received (left) and powdered chitin

Table 2.1 Physical properties of chitin

Property Specification

Appearance Off-white flake material

Moisture 10%

Ash content 1.8%

Protein 2%

Iron content 2 ppm

(Adapted from material testing document provided by N.G. Alexander and Co. Pty

Ltd., Victoria, Australia 2009)

Microcrystalline cellulose (MCC) was obtained from Sigma-Aldrich (particle

size~20µm, 310697). Flex-262 was obtained from Kingfa Sci. & Tech. Company Ltd.,

Guangzhou, China 510520. PBSA (Trade name 1803F) and PBAT (Trade name 2003F)

were supplied by Zhejiang Hangzhou Xinfu Pharmaceutical Co., Ltd, China 311300.

Flex 262 was purchased from Kingfa Sci. and Tech. Co. Ltd., Guangzhou, China

510520. Polyethylene (PE) in pellet form was supplied by Allied Colours and Additives

Pty Ltd. (Mitcham, Victoria, Australia).

22

2.2 Constitution of chitin biocomposites

2.2.1 Chitin/LDPE biocomposites

Three blends of chitin containing additives (powder form) which contained varying

proportions of chitin, microcrystalline cellulose (MCC), nanoclay and calcium

carbonate were manufactured and supplied by MM Foods Pty Ltd (the exact proportions

of these blends are unknown as they are proprietary information of MM Foods Pty.

Ltd). The powdered blends were dried at 105°C overnight and mixed with stearic acid

(1%) and calcium stearate (1%) to make them suitable for pelletizing. Pelletizing was

performed at Allied Colours Pty Ltd. Pellets were made using a compression technique.

The three types of pellets were melt-blended with LDPE.

In order to make films, three different percentages of pellets (25%, 33% and 50%) were

used in combination with LDPE. The resulting compositions of the extruded films are

summarized in Table 2.2.

Table 2.2 Composition of Biopla plastic films

Name Composition

Biopla1-25 Chitin additive 1(25%) + LDPE (75%)

Biopla1-33 Chitin additive 1(33%) + LDPE (66%)

Biopla1-50 Chitin additive 1(50%) + LDPE (50%)

Biopla2-25 Chitin additive 2 (25%) + LDPE (75%)

Biopla2-33 Chitin additive 2 (33%) + LDPE (66%)

Biopla2-50 Chitin additive 2 (50%) + LDPE (50%)

Biopla3-25 Chitin additive 3 (25%) + LDPE (75%)

Biopla3-33 Chitin additive 3 (33%) + LDPE (66%)

Biopla3-50 Chitin additive 3 (50%) + LDPE (50%)

23

2.2.2 Chitin-Flex262-LDPE biocomposites

Flex 262 (a commercially available partially biodegradable polymer) was melt blended

with chitin (10%), cellulose (10%) and/or LDPE (40%) to produce films of varying

compositions (Table 2.3) which were subsequently utilized for biodegradation testing.

Table 2.3 Composition of EC films

Films Composition Thickness

EC1 Flex-262 50 µm

EC2 Flex262 + 60%LDPE 50 µm

EC3 Flex262 + 40%LDPE + 20% (MCC + chitin) 30 µm

2.2.3 Chitin-MCC-PBSA/PBAT biocomposites

Chitin and MCC were combined with PBSA/PBAT in varying proportions to make

composite pellets. The distribution of the different components in the composites is

given in Table 2.4.

Table 2.4 Composition of chitin-MCC-PBSA/PBAT composites

Name Chitin (%) MCC (%) PBSA (%) PBAT (%)

PBAT - - - 100

PBSA - - 100 -

PBAT-chi/MCC 15 15 - 70

PBSA chi/MCC 15 15 70 -

24

2.2.4 Chitin-PBSA/PBAT biocomposites

Likewise chitin composites were manufactured with PBSA and PBAT with the

exclusion of MCC (Table 2.5).

Table 2.5 Composition of chitin-PBSA/PBAT composites

Name Chitin (%) PBSA (%) PBAT (%)

PBSA 0 100 0

PBSA F10 10 90 0

PBSA F20 20 80 0

PBAT 0 0 100

PBAT F10 10 0 90

PBAT F20 20 0 80

2.3 Masterbatch and blown-film extrusion of chitin biocomposites

The general trend of preparing biocomposites for use in mechanical testing and/or

degradation studies involved drying the individual components (synthetic polymers,

chitin, MCC) to remove moisture in order to avoid possible hydrolysis degradation of

polymers and reduce unwanted voids in the produced samples (Wu et al. 2011; Shibata

et al. 2002; Averous and Boquillon 2004). The composites were directly compounded

using a co-rotating twin-screw extruder with a screw diameter of 4mm. Materials

extruded in the form of a thin rod and were subsequently pelletized into fragments

approximately 4mm long. The pelletized Biopla samples and EC1/EC2/EC3 samples

were further processed into blown films (25-50µm thick). In case of chitin-cellulose-

PBSA/PBAT and chitin-PBSA/PBAT samples, raw materials were fed into the extruder

using a feeder with a 50-60 rpm screw speed with a barrel temperature of 80°C. The

different zones and their temperatures were 80-100-120°C (consistent with processing

temperatures of PBSA and PBAT). In case of Biopla samples, an extrusion temperature

of 180°C was used (consistent with processing temperature of LDPE).

25

2.4 Characterization of chitin biocomposites

2.4.1 Test specimens

Pellets (approx. length: 4mm, diameter: 1mm) were used as respirometric test

specimens for chitin-cellulose-PBSA/PBAT samples. Blown films of Biopla and

EC1/EC2/EC3 samples (cut into 2×2cm pieces) were used for respirometric analysis.

Respirometric test specimens for chitin-PBSA/PBAT were made by compression

moulding (at 120°C for 5 minutes) and then cutting the moulded sheets into 2×2cm

pieces. Specimens for tensile strength testing were made for chitin-PBSA/PBAT and

chitin-cellulose-PBSA/PBAT samples according to ASTM D638 (2010) Type I

guidelines (by compression moulding at 120°C for 5 minutes).

2.4.2 Tensile Test

Ultimate Tensile strength and tensile modulus were determined for all formulations

using an Instron universal testing machine (series 4000) and tested in accordance with

ASTM D638-2010 (Type 1) at a crosshead speed of 50mm/min and load weight of

2kN. A minimum of five specimens per each formulation were tested until fracture,

from which a mean and standard deviation were calculated. Bluehill® software was used

for all calculations.

2.4.3 Notched Izod Impact Test

The experiments were carried out using an Izod Impact Tester (Model: IT612,

Davenport Ltd, England). Notched specimens for impact tests according to ASTM

D256 (2010) were cut from the molded plaques and were notched using a bench-top

milling machine. A minimum of 5 specimens with notch length of 1mm were tested for

each sample and the results were averaged for each sample.

26

2.4.4 Scanning Electron Microscopy (SEM) of composites

Surfaces of biodegraded samples and the fracture surfaces of the impact specimens were

mounted onto SEM stages with double-sided conductive tape and then coated (to

approximately 300Å) with gold prior to examination. Samples were viewed under

different magnifications by the ZEISS SUPRA 40 VP field emission scanning electron

microscope (SEM) with INCA 250 energy dispersive X-ray spectrometer (EDS) system.

2.4.5 Fourier Transform Electron Microscopy (FTIR)

Surfaces of neat and biodegraded samples were scanned using a Spectrum One® FT-IR

Spectrometer (Perkin Elmer Inc.). Film samples (EC1/EC2/EC3) were inserted into a

holder (supplied with instrument) and placed in the path of the laser beam. The

frequencies were corrected for background noise and data were analysed by Spectrum

3.02.01 software. The FTIR spectra were recorded at a resolution of 2cm-1 and an

accumulation of 50 scans. The wavelength range was 450-4000 cm-1.

2.5 Biodegradation of chitin composites

2.5.1 Compost

For biodegradation studies of Biopla composites, 2-3 month old mushroom compost

was obtained from CERES organic farm (Brunswick, Victoria, Australia). For all other

biodegradation studies 2-3 month old compost was kindly donated by Natural Recovery

Systems (Dandenong South, Victoria, Australia). The organic compost had been

prepared from food-processing waste, supermarket produce waste, sawdust and

shavings, grass clippings, tree pruning, waste fibre and sewage sludge. Before use, the

compost was sieved through a brass sieve of 6.10 mm aperture and large pieces of glass

and stone were manually removed. The typical characteristics of the compost were: C/N

ratio, 20-30; volatile solids, 40%-50%; moisture content, 50-55%; pH, 7.5-8.0 (data

provided by supplier, pH and moisture content were measured after purchase to verify

supplied data).

27

2.5.2 Bioreactors

Glass bioreactors were used composting. They were filled with: compost only (blank),

compost + cellulose powder (positive reference) or compost + composite specimens

(test material).

2.5.3 Composting conditions

Composting of polymers was done according to AS ISO-14855 (2005). The compost to

polymer ratio was adjusted to 6:1 (dry weight basis) and moisture was maintained at 50-

60%. A constant temperature of 58°C was maintained throughout the experiment. Air

was flushed into the reactors at a rate of 100-150 mL/min and the content of CO2 in the

outgoing air was measured at least 4 times a day using an infrared CO2 meter (ADC

2000 series gas monitor). The measurement of CO2 was automated and the complete

description of the apparatus and analytic system is described elsewhere (Jayasekara et

al. 2001). MCC was used as a positive control (AS ISO14855-2005). A carbon mass

balance calculation was performed which gave the true biodegradation of the polymers

(equation1). During aerobic biodegradation, the polymer produces CO2, microbial

biomass, water and mineral salts as end products of bio-oxidation. CO2 evolved is a

function of the amount of carbon in the polymer and its measurement gives the rate of

biodegradation of the polymer in compost.

)(

)()((%)

,2

,2,2

MATERIALTEST

COMPOSTMATERIALTEST

COvollTheoretica

COvolCumulativeCOvolCumulativenDegradatio

Equation 1: Equation for calculating the percentage degradation of test material with

time.

Table 2.6 Weight of Sample, MCC and Compost for different composting tests

Biocomposite type Sample (g) MCC (g) Compost (g)

Chitin-Flex262-LDPE biocomposites 100 100 600

Chitin-MCC-PBSA/PBAT biocomposites 53 53 318

Chitin-PBSA/PBAT biocomposites 50 50 300

28

Figure 2.3 Photographs of the respirometric unit showing set-up and direction of airflow

2.5.4 Organic Carbon analysis

Total organic carbon was determined using a Leco® CN-2000 (serial no. 3396) CHN

analyser. The instrument was calibrated with an EDTA Leco® standard with a carbon

content of 40.94% and a nitrogen content of 9.56%. The total inorganic carbon content

was determined by measuring the quantity of ash produced after exposure to 550C in

accordance with manufacturer’s instructions.

29

2.6 Molecular techniques for evaluation of microbial populations

2.6.1 Buffers and solutions

All buffers and solutions (Table 2.6) were prepared with sterile MilliQ water (Millipore)

in accordance with to Sambrook and Russell (2001) unless otherwise indicated and

were autoclaved (121°C for 20 min) before use. All chemicals and reagents used to

prepare buffers and solutions were of analytical quality.

Table 2.7 Buffers and solutions used for nucleic acid extraction and agarose gel electrophoresis

The solutions required for DNA sequencing (Table 2.8) were prepared according to

instructions provided by AGRF (Australian Genome Research Facility, Melbourne,

Australia).

Table 2.8 Buffers and solutions used for DNA sequencing

Buffer/Solution Composition

BDT reaction buffer, 5X 40mM Tris pH 9.0, 10mM MgCl2

MgSO4 stock solution 0.2 mM in 70% ethanol

Buffer/Solution Composition

TE buffer 10mM Tris, 1mM EDTA

TAE buffer (Tris-acetate EDTA) buffer,

50X

Tris base, 6.5 M EDTA disodium salt,

pH 8.0

Agarose gel electrophoresis loading dye 30% (v/v) glycerol, 1 mg/mL xylene

cyanol, 1 mg/mL bromophenol blue

Sodium Acetate (NaAc) 3M, pH 6.0

Extraction buffer 50mM Tris pH 7.6, 50mM EDTA, 5%

Sodium Dodecyl Sulphate (SDS)

30

The media used for culturing bacteria and fungi were prepared according to Sambrook

and Russell (2001) and are listed in Table 2.9. All media and solutions were prepared

with distilled water and autoclaved/filter sterilized.

Table 2.9 Solutions and media for cultivation of bacteria and fungi

Solution/medium Composition

Ampicillin 20 mg/mL

IPTG (isopropyl-β-D-thiogalactopyranoside) 0.1 M

Luria broth (LB)

10 g/L tryptone, 5 g/L yeast extract,

5 g NaCl, 15 g/L agar (for plates

only)

SOC media (Super Optimal broth

with Catabolite repression)

0.5% yeast extract, 2% tryptone, 10

mM

NaCl, 2.5 mM KCl, 10 mM MgCl2,

10 mM MgSO4, 20 mM glucose

X-gal (5-bromo-4-chloro-3-indolyl-β-D-

galactopyranoside)

5% (w/v) in dimethylformamide

31

2.6.2 Isolation of total DNA from compost

DNA was extracted from compost samples after the end of composting trial using the

following protocol. Briefly, 1 mL of extraction buffer and 0.15g of compost sample

were added to a 1.5 mL microcentrifuge tube containing 0.4-0.5 mL of glass beads

(0.10mm diameter). The contents were thoroughly homogenised with a Fast-Prep®

instrument for 30 seconds at 5.5 ms-1. This was followed by centrifugation for 3 minutes

at 14,000 x g and then the supernatant (approx. 900 µl) was decanted into a fresh tube.

A half volume of phenol/chloroform/isoamyl alcohol was added and tube was gently

inverted until the solution became milky. Then the tube was centrifuged for 3 min at

14,000 x g until the phases were well separated. The aqueous phase was gently removed

and an equal amount of chloroform was added to it and centrifuged as above. The

aqueous phase was transferred to a new tube. Nucleic acids were then precipitated by

adding 0.1 volumes of 3M Sodium acetate solution and 0.7 volumes of isopropanol. The

tube was inverted to mix well and kept at room temperature overnight. DNA was

precipitated by centrifugation at 14,000 x g for 30 min at 4°C. The resultant dark brown

pellet was washed with 0.5mL ice-cold 70% ethanol and centrifuged for 5 mins at 4°C.

The residual ethanol was carefully removed and the pellet was allowed to dry for 5 mins

at room temperature. Subsequently, 50 mL of sterile TE was added to the tube and the

pellet was dissolved by gentle tapping.

2.6.3 Agarose Gel Electrophoresis

For quantification of DNA and to analyse sample integrity, agarose gel electrophoresis

was used. Agarose gels (1.0% w/v) were prepared in 1X TAE buffer, with 0.5 μg/mL

ethidium bromide added to the cooled gel solution (Sambrook and Russell, 2001).

Electrophoresis was carried out at 100 V for 60 min. DNA samples were mixed with a

6X loading dye. A molecular weight marker, Hyperladder TM 1 (200-10,000 bp;

Bioline), was used to estimate the size of DNA bands. DNA concentrations were

determined by comparing the intensity of the bands with the intensity of the marker

fragments containing known amounts of DNA. The gels were viewed under a UV

transilluminator and photographed (Chemidoc XRS Documentation Station, Bio-Rad).

32

2.6.4 Polymerase Chain Reaction

The crude DNA preparations were separated from associated humic acids and other

contaminants by agarose gel extraction using Zymoclean™ Gel DNA recovery kit

(Zymo Research Corporation, USA) The purified extracts were diluted 1:10 and used as

a template for PCR. The bacterial 16S ribosomal RNA region (Schabereiter-Gurtner et

al. 2001) was amplified with bacterial primers 27F (5′ AGA GTT TGA TCC TGG CTC

AG 3′) and 907R (5′ CCG TCA ATT CMT TTG AGT TT 3′) purchased from Sigma-

Aldrich Co. Ltd. 25µl PCR reactions containing 12.5µl Biomix™ 2X (contains

BIOTAQTM DNA Polymerase, (NH4)2SO4, Tris-HCl, Tween 20, dNTPs, MgCl2;

Bioline, Alexandria, Australia), 1 µl DNA and 1 µl of each primer were prepared. The

PCR conditions were as follows: 95ºC- 10 minutes (initial denaturation), 30 cycles of

94ºC- 1:00 min (denaturation), 55ºC- 1:00 min (annealing) and 72ºC- 2:00 min

(extension), followed by a final extension of 72ºC- 10 minutes. Five μL aliquots of the

PCR products were mixed with 6X loading dye and electrophoresed as above.

2.6.5 Cloning of PCR fragments

The PCR fragments were purified using the Promega PCR purification kit according to

the manufacturer’s instructions and ligated into the pGEM®T Easy (Promega,

Alexandria, Australia) vector following the manufacturer’s protocol. E. coli JM109

High efficiency competent cells were prepared using the Inoue method (Inoue et al.

1990) and were used for transformation. Transformed cells were plated on Luria-Bertani

agar supplemented with ampicillin (100µg/mL), X-gal (0.5mM) and IPTG (80µg/mL)

and incubated overnight at 37°C. Colonies which were white in colour were identified

as containing inserts. The colonies were individually picked, and grown overnight in 5

mL of LB broth supplemented with ampicillin. The vector was purified from the E. coli

cells using Plasmid miniprep kit (Promega, Alexandria, Australia) according to

manufacturer’s instructions. The isolated plasmids were sequenced using T7 (Forward,

5’ GTAATACGACTCAGGGC 3’) and SP6 (primers, 5’ TTTAGGTGACACAGAATC

3’) primers.

33

2.6.6 Sequencing

Restriction digestion of the plasmids was performed with EcoR1. The presence of

inserts was confirmed by agarose gel electrophoresis. The plasmids containing the insert

were sequenced using Applied Biosystems PRISM™ Big Dye Terminator Mix-version

3.1 and comparative analysis was performed using basic local alignment search tool

(BLAST) to compare these sequences to those available in the GenBank database. This

provided information on the identities of gene sequences and provided an estimation of

the genetic diversity in the examined samples.

2.7 Statistical Analysis

Student’s T-test was used to analyse statistical significance of test results. Null and

alternative hypothesis were tested using 2 tailed distributions and 2 sample unequal

variance method.

34

3. MECHANICAL PROPERTIES OF CHITIN

COMPOSITES

3.1 Introduction

Natural fibres such as chitin have a number of advantages over glass fibres, mainly: (i)

their low density (which makes it possible to obtain lighter composites), (ii) their

renewable character, (iii) their ubiquitous availability (iv) cheap cost and (iv) their low

abrasiveness which ensures minimum damage to the processing tools (Mohanty et al.

2000). The mechanical properties of a polymer blend or composite strongly depend on

its composition, interfacial adhesion and morphological structure. In most composite

materials, effective wetting and uniform dispersion of the different phases of the

composites are required to obtain a composite with satisfactory mechanical properties

(Baiardo et al. 2004 and Wu 2010). In this study, the effect of chitin addition and

chitin+MCC addition into two different polymers, PBSA and PBAT, on the mechanical

properties of the polymers was investigated.

3.2 Results and Discussion

3.2.1 Tensile test

The tensile strength and modulus of polymers and composites were determined using

specimens prepared as depicted in Fig. 3.1

35

Figure 3.1 Typical specimens for impact test (top) and tensile test

Table 3.1 shows a comparison of the tensile modulus and tensile strength of PBSA and

its composites. It can be seen that addition of 10wt% of chitin to PBSA did not result in

significant reduction of tensile strength (p value 0.23) of PBSA but resulted in

considerably significantly higher modulus (+60%, p value 0.0007). However when

20wt% chitin was added to PBSA, there was a significant drop in tensile strength (p

value 3.5×10-7). The decrease in tensile strength (-20%) and accompanying increase in

tensile modulus (+150%) was almost linear in this instance (Table 3.1). In the case of

PBAT, the decrease in tensile strength was greater due to addition of fillers when

compared to PBSA as demonstrated by the 21% and 39% decrease in tensile strength

for PBAT10 and PBAT20, respectively. The reduction of tensile strength in the PBAT

composites was significant for both 10wt% and 20wt% chitin filled composites (p value

0.03 and 0.0019 respectively) when compared to PBAT. In contrast significant

increases in modulus were observed for the 20wt% chitin filled composites (p value

0.007) when compared to PBAT. Increase in modulus was however found to be non-

significant in case of 10wt% chitin filled PBAT composites.

36

Table 3.1 Tensile strength and modulus of chitin filled PBSA and PBAT composites at different filler loadings

Composite

Description

Tensile Strength at break

(MPa)

Modulus of elasticity

(MPa)

Notched Izod

Impact Strength

(J/m)

Mean SD Change Mean SD Change Result Change

PBSA(1803F) 18.39 0.37

313.5 11.58 104

PBSAF10 16.81 2.49 -9% 501.9 34.51 60%

49

-53%

PBSAF20 14.78 0.38 -20% 786.4 52.95 151% 44 -58%

PBSA-

CHI/MCC 7.8 1.05 -58% 998.6 111.02 219% 18 -83%

PBAT(2003F) 9.85 1.14 12.7 2.94 - -

PBATF10 7.79 1.22 -21% 12.2 1.66 -4.4% - -

PBATF20 6.00 0.26 -39% 154.2 2.75 1110.4

% 98 -

PBAT-

CHI/MCC 5.66 0.17 -43.5% 181.3 11.06 1386% 51 -

37

Figure 3.2 Typical Stress-stain curves for 1803F (PBSA) and its composites

Figure 3.3 Typical Stress-stain curves for 2003F (PBAT) and its composites

-5

0

5

10

15

20

25

30

0 10 20 30 40 50 60

Ten

sile

Str

ess

(M

Pa)

Tensile Strain (%)

PBSAF

PBSAF10

PBSAF20

PBSA CHI MCC

-2

0

2

4

6

8

10

12

14

0 100 200 300 400

Ten

sile

Str

ess

(M

Pa)

Tensile Strain (%)

PBATFA

PBATF10

PBATF20

PBAT CHIMCC

38

The shape of the stress–strain curve is affected by the addition of fillers (Mohanty

2000). For unblended pure polymers, the behaviour is similar to that of ductile

polymers. The stress-strain curves of the PBSA/PBSA composites and PBAT/PBAT

composites (Fig. 3.2 and Fig. 3.3) show a typical tensile behaviour of polymer

composites that are made of a rigid dispersed phase in a soft matrix. The tensile strength

of chitin-filled biocomposites decreased along with increasing filler content. The

deformation mechanism of polymer with fillers depends on the relationship between the

strength and the yield stress of the unfilled polymer (Correlo 2005). According to

Bazhenov (1995), if the strength of the unfilled polymer is lower than its yield stress, an

increase in particle content leads to the formation of a brittle composite. This would

appear to be the case for both PBAT and PBSA composites where failure occurs at low

strains, and the material fails immediately after reaching maximum stress. This proves

weak interfacial adhesion between the filler and the matrix, which promotes micro-

crack formation at the interfacial area. This trend in biocomposites has been

documented by a number of authors (Mani and Bhattacharya 2001; Correlo 2005;

Bazhenov 1995) and the general reasoning has been the poor compatibility of polar,

hydrophilic natural fibres and non-polar hydrophobic PBSA/PBAT matrix.

The reinforcement effect however could be observed as an increase in stiffness

(modulus) of the composites with increasing filler loading which suggests sufficient

stress-transfer across the matrix-filler interface. The exception was observed in the case

of PBATF10 where the non-significant difference in modulus when compared to PBAT.

On the other hand, the increment in modulus for PBAT20 was significantly higher (p

value 8.5×10-13) and was almost 13 times (+1110%) that of virgin PBAT and this

magnitude of increase was exceptionally higher when compared to modulus increase in

case of PBSA composites. This indicates that the 20wt% filler greatly reduced PBAT

ductility/elongation and increased its brittleness and stiffness. Correlo et al. (2005) have

reported that when a relatively softer matrix is reinforced by a high modulus filler, the

polymer next to the filler particle is highly restrained which enables a major portion of

the load to be carried by the filler. In this case, PBAT is a much softer polymer and

shows extensive elongation when compared to PBSA (Fig. 3.2 and Fig. 3.3). Therefore,

much of its stress is immediately transferred to its fillers.

39

In the literature, the change in tensile strength of the composites has been reported as a

loss in strength (Mishra et al. 2002), no significant change (Mohanty et al. 2000) or

improved strength (Mishra et al. 2002) compared to the composite containing either

untreated fibres or equivalent pre-treated fibres. As explained above, if the filler

concentration exceeds the maximum packing fraction, particle interaction will result in a

higher stress concentration and earlier breakage of the composite. Another reason for

the inferior tensile performances of chitin as filler (with the exception of PBSAF10)

compared to other reported commercial fillers may be the irregular shape of its particles

(Mohanty et al. 2000). For irregularly shaped fillers, the strength of the composites is

lower as the filler carries less stresses than regular filler.

Ashori and Nourbakhsh (2010) found that addition of 8wt% of MCC to

polypropylene/wood flour composites significantly enhanced the tensile strength of

composites when compared to those without MCC. Thus, it was expected that addition

of MCC would result in better interaction between the filler and the matrix. In PBAT-

CHI/MCC (30wt%), some reinforcing effect could be observed since the drop in tensile

strength was comparatively low (5%) when filler loading was increased from 20wt% to

30wt% compared to substantial losses in tensile strength (18%) when filler loading

increased from 10wt% to 20wt%. However, in the case of PBSA-CHI/MCC (30wt%)

composites, the decrease in tensile strength (58%) was significantly higher than

expected when compared to PBSA. Also, the increase in tensile modulus was less than

expected (non-linear). Therefore, MCC was able to provide some degree of

reinforcement to PBAT but not PBSA. The reasons for this behaviour could be

attributed to inefficient dispersion and incompatibility between MCC and the PBSA that

lead to formation of MCC aggregates (Mathew et al. 2005).

40

The results presented above provide comparison between untreated fibre composites

and neat polymer. With the exception of PBSAF10, reduction in the tensile strength due

to chitin and chitin/MCC indicates that chitin is not competitive with reinforcing fillers.

The decrease in strength and the increase in stiffness with increase in filler loadings

only make chitin and MCC commercially attractive in terms of cost reduction rather

than mechanical reinforcement. In the literature, most improvements in composite

fabrication and mechanical properties are attributed to improved fibre-polymer

interaction due to fibre treatments that increase surface roughness of the fibre (Hill and

Abdul Khalil 2000; Razera and Frollini 2004) and/or dissolution of surface waxes and

increasing hydrophobicity. Therefore, treatment/compatibilization of chitin fibres and

optimization of processing conditions can lead to composites with improved mechanical

performance.

3.2.2 Impact Strength

The impact strength decreased in PBSA composites by 53%, 58% and 83% with

increasing filler loading of 10wt%, 20wt% chitin and 30wt% chitin/MCC, respectively.

Similar findings were observed by other researchers where impact strength was reduced

upon addition of fillers to PBSA (Tserki et al. 2003). These results show a similar trend

to the tensile strength of the composites. Both properties are sensitive to embrittlement

of the matrix as the tensile strength is a measure of resistance of the composite material

to a gradually applied load and notched impact strength is measure of the response of

the material to a suddenly applied load. In an impact sample, stress is concentrated at

the tip of the notch. The behaviour is then controlled by propagation of the crack,

whereas in tensile strength, the force applied causes a stress in the sample which is

transferred through the phases, the stress concentrates at the point where the phases

become discontinuous thereby causing a crack (Mohanty et al. 2000). The crack

propagates due to further stress (Adhikari et al. 2003). Both these properties are also

influenced by the level of bonding between the filler and matrix, as explained above.

Poor interfacial bonding induces micro-spaces between the filler and the matrix

polymer, and these cause more micro cracks when impact occurs, which allows crack

propagation to occur more easily (Adhikari et al. 2003). The decreased impact strength

of the composite observed here is therefore indicative of weak interfacial bonding

between the chitin/MCC and PBSA/PBAT, attributable to the -OH groups of fillers

used.

41

3.2.3 Composite morphology

Compression molded samples were found to show skin core morphology. Chitin

domains tended to be enclosed in the inner regions of moldings, confirming the

previous reports that the continuous phase consists of polyester and the dispersed phase

is composed of chitin domains.

To evaluate the composite morphology, SEM was employed to examine tensile

fractures in the surfaces of PBSA and PBAT neat samples as well as composites (Fig.

3.4 and Fig. 3.5). The SEM microphotographs show that the polymer-filler interfaces

are discontinuous and are coupled with fibre pull-outs, cracks and micro cavities. In

case of PBSAF10, however, the presence of such failures is observably less and this is

reflected in the non-significant change in tensile strength when compared to pure PBSA.

In accordance with Wu et al. 2012 this may be due to the lesser amount of filler (10%)

in that composite.

42

43

Figure 3.4 SEM characteristics (magnification 200X) of 1803F (PBSA) and its composites after tensile fracture A) Neat PBSA B) PBSA-CHI/MCC C) PBSAF10 D) PBSAF20

44

45

Figure 3.5 SEM characteristics (magnification 200X) of 2003F (PBAT) and its composites after tensile fracture A) Neat PBAT B) PBAT-CHI/MCC C) PBATF10 D) PBATF20

46

4. BIODEGRADATION OF CHITIN

COMPOSITES

4.1 Introduction

Bioremediation is a grouping of technologies that use microbiota (typically,

heterotrophic bacteria and fungi) to degrade or transform hazardous contaminants to

materials such as carbon dioxide, water, inorganic salts, microbial biomass and other

by-products that may be less hazardous than the parent materials. Biological treatment

has been a major component for many years in the treatment of municipal and industrial

wastewaters (EPA, 1998a; EPA, 2000). In recent years, biological mechanisms have

been exploited to remediate plastic wastes from water and soils. Biodegradable and

compostable polymers have garnered attention from industries, consumers and

governments as a potential way to reduce municipal solid waste, since they can be

recycled or have energy recovered through composting into soil amendment products.

For such novel materials to make claims to be “environmentally friendly”, it must be

proven, using scientifically based and accepted methods, that they undergo

biodegradation under natural environmental conditions, such as in landfills and

compost. According to AS ISO 14855 (2005) and ASTM D5338 (2003), composting is

a process where biodegradable materials undergo biological degradation to yield carbon

dioxide, water, inorganic compounds and biomass, at a rate consistent with other known

compostable materials such as cellulose, and leaving no visually distinguishable or toxic

residues.

PBAT and PBSA are globally certified biodegradable thermoplastic derived from fossil

fuels. They are ideal for disposable packaging as they decompose readily in compost.

However, as the price of crude oil increases, the use of PBAT and PBSA also faces a

challenge. In recent years, materials called biocomposites, which are composed of

agricultural residues/waste materials and biodegradable polymers, have become very

attractive materials in terms of their properties and biodegradability after their uses.

47

Chitin is an abundantly available structural polysaccharide found in crustaceans and

insects. The haemostatic properties of chitin have made it an ideal coating for

commercially available medical sutures. Chitin is also anti-microbial making it

applicable for food processing and preservation. However, scant information exists in

relation to the biodegradation properties of chitin-LDPE/polyester composites. This

chapter details studies that were undertaken to investigate the biodegradation of melt-

blended chitin composites. The effect of chitin addition (small and large amounts) as

well as the addition of MCC on the aerobic biodegradability of the LDPE/polyester

based composites is also investigated. Additionally, the effect of chitin-based plastics on

the bacterial population of the compost has also been evaluated.

4.2 Results and Discussion

4.2.1 Biodegradation of chitin/LDPE composites-Test 1

MM Foods Pty Ltd, Bayswater, Victoria has designed a unique method of blending

chitin along with other biodegradable materials and polyolefin that can be converted

into a range of product for different applications. Finely ground chitin was melt-blended

along with low density polyethylene (LDPE) to produce thin films (Fig. 4.1). In a

preliminary study, the susceptibility of these composite films to biological degradation

was analysed.

Weight loss of the proprietary composites (containing different concentrations of chitin

and cellulose along with polyolefin) was determined after burying 5cm×7cm strips of

plastics in 300gms of compost contained in glass beakers. Constant temperature was

maintained at 48±3 ° C. Water was added regularly to compensate loss and moisture

content was measured regularly with a Sartorius MA 100™ Moisture analyser. The

moisture content was kept to 50-60%. The rate of degradation was compared to

commercially available biodegradable plastic bags (starch-based) and low density

polyethylene (LDPE) bags. After 4 weeks the strips were recovered, washed carefully

and the weight losses were determined.

48

a)

b)

c)

Figure 4.1 (a, b and c): Biopla composites (test materials for soil-burial study)

49

Results showed that the blend containing approx. 25% of chitin underwent substantial

weight loss (6.86%) compared to pure LDPE (0.1%).

This primary investigation indicated that the LDPE-chitin blend had higher rates of

disintegration in compost compared to pure LDPE.

4.2.2 Biodegradation of chitin-Flex262-LDPE composites-Test 2

Chitin-Flex262-LDPE composites were tested for biodegradation under composting

conditions as discussed in Chapter 3.

The moisture content of the compost was 55% and pH was found to be 8.9. The

parameters to assess the validity of the test were evaluated and found to meet the

requirements of AS ISO 14855 (2005) (Table 4.1).

Table 4.1 Validation criteria for AS14855

Validation Criteria Results AS ISO 14855

(2005)

Requirements

Degree of biodegradation of the reference

material (cellulose) at 45 days

81 10.4% (>70%)

Passed

Difference in biodegradation between the

reference material (cellulose) vessels at test end

7.1% Std Dev (<20%)

Passed

Blank inoculum (compost) production of CO2

per gram of solid volatiles in 10 days

71mg (50<150mg)

Passed

50

Finely ground chitin and cellulose could be melt-blended along with aliphatic polyesters

and low density polyethylene (LDPE) to produce thin films.

Figure 4.1 Chitin-Flex262-LDPE composites (test materials for composting study)

These films were found to be susceptible to microbial degradation under composting

conditions. The commercially available biodegradable blend (Flex-262) film (EC1) was

found to degrade 40+0.4 % in 6 months. EC3, which contained chitin and cellulose,

degraded by 22 +5.2% and EC2 did not show any degradation (Table 4.2). The

complete absence of biodegradation in EC2 films could be attributed to the thickness of

the film which was found to be 20µm more than EC1 and EC3. Overall, the degradation

rate of the EC3 composites was slow and the composites did not meet the ASISO14855

criteria for biodegradable plastics. This can be attributed to the presence of LDPE which

has been shown to be resistant to microbial attack (Kalra, 2002).

51

Table 4.2 Percentage biodegradation of Chitin-Flex262-LDPE composites.

Materials Percentage biodegradation

after 45 days of compostinga

Cellulose 85.1+7.1

EC1 40.1+0.4

EC2 NONE

EC3 22.6+5.2

a Values are average ± SD of triplicate trials

Figure 4.2 Graph showing percentage mineralization of Chitin- Flex262-LDPE composites

-20

0

20

40

60

80

100

0 50 100 150 200

Perc

enta

ge b

iodegra

datio

n

Time (days)

CELLULOSE

EC1

EC3

EC2

52

Figure 4.3 Photographs depicting changes in appearance of films after composting

These composites were further characterized by scanning electron microscopy (SEM)

using a ZEISS SUPRA 40 VP field emission scanning electron microscope

53

Figure 4.4 Scanning electron microscopy of composite films (300 X magnification)

The FTIR data (Fig. 4.6) obtained using a Perkin Elmer Spectrum One clearly showed

that, in EC3 composites, the ester groups of the polyester and alcohol groups of

cellulose, chitin and Flex-262 were affected rather than carbonyl groups of LDPE.

Flex-262 is a commercially available biodegradable polymer which was utilized to

impart biodegradability to the LDPE-chitin blend in addition to maintaining

processability (into films).

54

Figure 4.5 FTIR Spectroscopy showing changes in EC3 after composting

In the last few decades, the usage of LDPE for packaging applications increased many-

fold because of its easy processability, light weight, low cost, good resistance to

chemicals and microorganisms, and good mechanical properties. However in recent

times, the modification of LDPE packaging has gained importance, due to its non-

biodegradability, leading to superior management of plastic waste nuisance.

Investigations of LDPE and starch composites have shown the composites to be

susceptible to photo degradation, natural weathering and soil burial. Gupta et al. (2007)

studied the biodegradation of LDPE blended with natural gum and found loss in tensile

strength of the composites after soil burial. Makarios Laham and Lee (1995) observed

the degradation of chitin filler in chitin-LDPE composites after soil burial and

enzymatic degradation; nonetheless, they could not demonstrate definitive evidence for

simultaneous degradation of LDPE. Studies of loss in tensile properties and crystallinity

of LDPE-starch composites by Elanmugilan et al. (2013) suggested rapid degradation of

starch during soil burial studies thereby leading to the development of cracks and holes

in the composites. However, there was no significant degradation of the LDPE portion.

The suggested reason is the lack of chain-scission of LDPE molecules due to the

absence of light and sufficient oxygen.

55

Natural weather ageing, which includes the above-mentioned conditions, leads to

significant degradation of LDPE in LDPE composites due to active oxidation and

exposure to wind and sand particles (Andrady et al. 1993; Al-Mafra 1998; Elanmugilan

et al. 2013). As mentioned earlier, the composites in this study could be processed into

blown films and demonstrated good mechanical properties (data not shown); however,

they were not fully degradable under composting conditions. Therefore, it is possible

that a combination of UV treatment/natural weathering and composting can be a

solution to achieve complete degrade such composites/composites.

56

4.2.3 Biodegradation of chitin-PBSA/PBAT composites–Test 3

Poly (butylene succinate-co-adipate) (PBSA) and poly (butylene adipate-co-

terephthalate) (PBAT) can be produced by polymerization of glycols with dicarboxylic

acids. The polyesters are well known for their excellent mechanical properties,

proccessability and high filler compatibility, and can be completely degraded within a

short period by biodegradation (Kim and Kim 2006). PBSA and PBAT have been used

in composites with other polymers as a packaging material and for planting cups and

disposable cups, and have been proposed for use in agricultural applications, including

plastic bags, transparent films for wrapping food, and mulch films (Chen and Zhang

2010; Olivato et al. 2012). However, their potential use is limited due to the high cost of

production. A range of waste products and agricultural residues have been used as fillers

to produce cost-effective composites of PBSA and PBAT. Attempts have been made to

convert materials such as rice husk, wheat bran, sugarcane bagasse and its composites

into industrially useful products (Wu 2012). However, very few attempts have been

made to combine chitin and MCC with either PBSA or PBAT in order to make

inexpensive biodegradable composites. According to my knowledge, this is the first

attempt to document biodegradability of such novel composites in accordance with

ASTM/ISO Standards.

Chitin-PBSA/PBAT composites were tested for biodegradation under composting

conditions as discussed in Chapter 2. The moisture content of the compost was 55% and

pH was found to be 8.9. The parameters to assess the validity of the test were evaluated

and found to meet the requirements.

57

Table 4.3 Validation criteria for AS14855

Validation Criteria Results AS ISO 14855 (2005)

Requirements

Degree of biodegradation of the reference

material (cellulose) at 45 days

76.46

6.35%

(>70%)

Passed

Difference in biodegradation between the

reference material (cellulose) vessels at test

end

6.35% Std.

Dev.

(<20%)

Passed

Blank inoculum (compost) production of CO2

per gram of solid volatiles in 10 days

92 mg (50<150mg)

Passed

Figure 4.6 Chitin-PBSA composites (test materials for composting study)

58

Figure 4.7 Chitin-PBAT composites (test materials for composting study)

Figure 4.8 Graph showing percentage mineralization of Chitin-PBSA/PBAT composites.

After a 15-day incubation period, visible macroscopic evidence of microbial growth

with gradual erosion and cracking was observed on the surface of the PBAT/PBSA and

composites. After 30 days, the disruption of the matrix structure became more obvious.

It was also observed that the PBSA and PBAT test materials underwent embrittlement

faster than the composites. This difference in degradation rate was confirmed by

increased percentage mineralization of PBSA/PBAT matrix with incubation time

compared to the composites. Table 4.4 illustrates the fact that the degradation rates of

the native polyesters were higher than those reinforced with chitin and the total

degradation decreased with increasing chitin content. It was also found that the presence

of chitin had a greater slowdown effect on PBAT when compared to PBSA. Although

these two polyesters are known to have similar biodegradation rates, it was found that

neat PBAT used in this study decomposed at a higher rate compared to neat PBSA

(Table 4.4 and Table 4.6). The slowdown effect caused by incorporation of chitin was

thus more prolific in PBAT composites.

-20

-10

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50

Perc

enta

ge b

iodegra

datio

n

Time (days)

Cellulose

2003F(PBAT)

PBAT F10

PBAT F20

1803F (PBSA)

PBSA F10

PBSA F20

59

Table 4.4 Percentage mineralization of Chitin-PBSA/PBAT composites.

Materials Percentage mineralisation after 45

days of compostinga

Cellulose 76.46+ 6.35

PBSA 29.79+ 3.41

PBSA F10 26.03+ 0.22

PBSA F20 23.77+ 4.87

PBAT 36.13+ 3.06

PBAT F10 30.18+ 4.62

PBAT F20 21.48+ 5.71 a Values are average ± SD of triplicate trials

The difference between percentage mineralization of PBSA composites were found to

be statistically non-significant when compared to pure PBSA. However the decrease in

the rate of mineralization in the composites when compared to pure PBSA, obtained in

this test can possibly be explained by unravelling some of the inherent properties of the

structure of chitin. Although chitin is a natural material and ultimately biodegradable

under natural conditions, one has to keep in mind that the structure of the chitin

molecule is quite complex when compared to cellulose, especially due to the presence

of acetamide groups in the position of hydroxyl groups found in cellulose at the C2

carbon which leads to a very complex process of enzymatic utilization (Keyhani and

Roseman, 1999). Chitin is abundant in seawater because it is an integral part of the

crustacean skeleton and studies have focussed on chitin cycling in marine environments.

It has been reported that, although chitin is degraded eventually, it is somewhat resistant

to bacterial attack and the hydrolysis rate of chitin by-products has been found to exceed

the mineralization rate of chitin (Kirchman and White, 1999). McCarthy et al. (1997)

suggested that chitin contributes to the pool for dissolved organic nitrogen in marine

environments and hence implied the slow release of chitin by-products. Reports of

chitin preservation in fossils (e.g. Stankiewicz et al. 1997) also suggest that chitin

degradation may be more complex than assumed.

60

Since there have been few studies of the biodegradation of chitin/readily degradable

polymer composites, the results obtained in this test as well as following tests were not

readily comparable. Wu (2005) studied the structure and properties of PCL

(polycaprolactone), which is a biodegradable polymer, and chitosan composites. These

composites were prepared using chitosan (a deacetylated form of chitin) that was

purified in the laboratory before use, melt-blended with PCL and prepared into 1mm

thick plates using a hot press. Biodegradation was tested using the soil burial method.

Exposure to the soil environment showed that weight loss in PCL-chitosan composites

were faster compared to native PCL. The author proposed poor compatibility between

the two phases to be the reason for higher weight loss in the composites. In the current

study, such a correlation between the composite strength and biodegradation was not

found. Although there are too many differing variables between the two studies which

limit the possibility of narrowing down the cause of conflicting results, an assumption

can be made considering the fact that chitosan used in the study by Wu (2005) was

chemically purified while the tests shown here used chitin which and not been

chemically modified or purified before processing. The purified chitosan may be prone

to higher enzymatic degradation compared to commercially available ‘chitin’ that was

used in this study.

PBAT and PBSA are designed to be fast degraders under composting conditions and the

data presented here seem to suggest that the composites containing chitin filler simply

could not maintain the same rate of degradation of neat polyesters in a 45 day

composting trial.

4.2.4 Biodegradation of chitin-MCC-PBSA/PBAT composites–Test 4

These were tested for biodegradation under composting conditions as discussed

previously. The moisture content of the compost was 59% and pH was found to be 8.9.

The reason behind the testing was two-fold. First, the aim was to quantify the time taken

to completely mineralize PBSA/PBAT composites and, second, the effect of adding

MCC to the composites on their biodegradation properties was assessed. The

parameters to assess the validity of the test were evaluated and found to meet the

requirements.

61

Table 4.5 Validation criteria for AS14855

Validation Criteria Results AS ISO 14855

(2005)

Requirements

Degree of biodegradation of the reference

material (cellulose) at 45 days

72.13 0.89% (>70%)

Passed

Difference in biodegradation between the

reference material (cellulose) vessels at test end

0.9% Std Dev (<20%)

Passed

Blank inoculum (compost) production of CO2 per

gram of solid volatiles in 10 days

112mg (50<150mg)

Passed

The melt-blended pellets were found to degrade significantly faster (p value 0.01) for

PBSA CHI/MCC when compared to PBSA native polyester pellets and after day 120

they were indistinguishable from the compost. In case of PBAT CHI/MCC the

difference in percentage mineralization after 120 days was non-significant when

compared to PBAT native polyester pellets and both were found to be indistinguishable

from the compost after test completion. The degradation rate could be divided into two

phases: an initial lag-phase where chemical hydrolysis of the polyesters occurred

followed by a phase of fast degradation. This is explained by the fact that chemical

hydrolysis of the polyesters lowers their molecular weight which initiates the enzymatic

degradation that is performed by the compost microflora. Both the composite pellets

and the native polyester pellets showed high rates of degradation under composting

conditions and can be regarded as fully biodegradable as they also met the criteria of

ISO 14855 of the degradation rate being within 90% of that of cellulose. In fact, it was

found that after the initial lag phase, the composites degraded at a rate faster than

cellulose. This can be due to the fact that the breakdown products of the PBSA/PBAT

fostered enumeration of specific bacteria which could rapidly increase their

mineralization and, thus, respiratory rate. This type of priming effect has been reported

previously in the case of glucose and starch amended composts (Shen and Bartha 1996,

Bellia et.al. 2000). During the experiment it was noticed that after 50-55 days (i.e. after

the rapid degradation phase of cellulose), the cellulose containing compost tended to

62

lose moisture more rapidly when compared to the test composts, although a uniform

moisturizing regimen had been observed throughout the experiment.

It is possible that the moisture holding capacity of PBSA/PBAT pellets, its composites

and breakdown products helped to maintain the moisture in the compost within the

bioreactors to a greater degree than cellulose and hence fostered higher respiratory

activity after the 50 day period. Being hydro-biodegradable, these materials tend to

absorb a lot of moisture before they start degrading. However, they do not degrade as

fast as cellulose, thereby withholding their water content till later stages.

Figure 4.9 Graph showing percentage biodegradation of Chitin-MCC-PBSA/PBAT

Table 4.6 Percentage mineralization of Chitin-PBSA/PBAT composites

-20

0

20

40

60

80

100

0 50 100 150

Perc

enta

ge b

iodegra

datio

n

Time (days)

Cellulose

1803F(PBSA)

PBSA BL

2003F(PBAT)

PBAT BL

Materials Percentage mineralisation after 120

days of composting

Cellulose 77.22 + 0.70

PBSA 73.63 + 5.59

PBSA BL 93.52 + 6.53

PBAT 84.92 + 1.65

PBAT BL 91.29 + 11.44

63

At first glance, it seems that the results obtained in Test 3 are in conflict with Test 4,

wherein the biodegradation rate of PBSA/PBAT in the former test was found to be

higher than the composites. It should be kept in mind that the polyester pellets (PBSA

and PBAT) in Test 4 did not undergo extrusion and were used as tests materials “as

received”. However, the composites (PBSA CHI/MCC and PBAT CHI/MCC) had been

melt-blended and extruded into pellets before use. For Test 3, it is to be noted that all

types of test materials underwent the same extrusion conditions (as explained in Chapter

2), unlike Test 4 and that lower molecular weight induces hydrolysis and the subsequent

breakdown process (Witt et al. 2001). Of course, another factor could be the presence of

MCC in the composites of Test 3. Composites with cellulose/cellulose derivatives have

been found to be more susceptible to biodegradation than native polyesters (Oldak et al.

2005, Wu 2012). The highest rate of degradation was observed in case of PBAT

composites followed by PBSA composites. Wu (2012) tested melt-blended maleic

anhydride-grafted poly (butylene adipate-co-terephthalate) (PBAT) and cellulose

acetate (CA) for biodegradability by soil burial and found that PBAT-CA composites

had a slightly higher rate of biodegradation when compared to PBAT. Similarly, Wu

(2012) found that when peanut husk (cellulose fibres) was used as a filler in PBAT (10-

40% by weight), the composites degraded at faster rate compared to native PBAT.

Thus, incorporation of MCC in chitin-polyester composites may lead to enhanced

biodegradation properties as compared to chitin-polyester composites. However, as

evidenced from Chapter 3, the incorporation of MCC leads to significant reduction of

composite strength. Thus, for commercial applications of the proposed composites, the

balance between mechanical properties and biodegradability of the composites should

be considered.

64

4.2.5 Microbiota associated with biodegradation of chitin-MCC-PBSA/PBAT

Microorganisms play a key role in recycling of biodegradable polymers. Identifying the

microbial species that are involved in active degradation and/or the enzymes involved

could help in selection of treatments for ultimate disposal/recycling of the polymers.

Kijchavengkul et al. (2010) assessed the biodegradability of PBAT films in three

different types of composts (food, yard and manure), according to the ASTM D5338

(2003), and found that the rate of biodegradation varied widely among the three. The

authors attributed this to a lack of consistency in the microbial populations of the above-

mentioned composts and strongly suggested the need for in-depth examination of the

major microbiota involved in mineralisation of such polyesters to address such

ambiguity in degradation rates. In the biodegradation studies performed in this thesis,

assessment of polymer degradation was performed either by gravimetric analysis

(weight loss) or respirometric measurement (total amount of CO2 evolved during the

biodegradation process). While these methods provide an indication of the

biodegradability of polymers under a specific set of conditions, they do not reveal any

information about the diversity of microbial communities which underpin the observed

biodegradation performance. Considering the great potential of PBAT/PBSA

composites in future commercial applications, identification of microbial species and/or

the enzymes promoting biodegradation of these new materials could assist in selection

of an efficient method for treatment or recycling at the end of their life cycle.

Since, less than 1% of bacterial populations can be cultured on synthetic media

preparations (Hugenholtz et al. 1998), the use of molecular techniques provide much

more reliable assessment of bacterial population in any environment (Amann et al.

1995).

65

In this project, molecular ecological techniques were used to assess the microbial

population changes in compost after incubation with the composites. This section

presents a first report on molecular investigation of biodegradation of PBAT composites

under aerobic composting conditions. Microbial community DNA was extracted from

degraded composite samples (Fig. 4.11). Small-subunit rRNA gene sequences (16S

rRNA) were amplified using polymerase chain reaction (PCR) (Fig. 4.12) and clone

libraries were generated.

The amplified cloned gene fragments were further sequenced and a comparative

analysis was performed using the basic local alignment search tool (BLAST) to

compare these sequences to those available in the GenBank database. This yielded

information on the identities of new gene sequences and provided an estimation of the

relative abundance of different micro-organisms in the compost environments.

66

Figure 4.10 Gel showing genomic DNA extracted from compost at Day 0 (Lane 1) and DNA ladder (Lane 2; Hyperladder I)

Figure 4.11 Gel showing PCR product of the expected size (~ 900 bp) for Day 0 compost in Lane 2. Lane 1 contains DNA Hyperladder I

1000

1500 bp

67

The microbial diversity in the blank compost samples was analysed to provide reference

information for detection of changes in the microbial population subsequent to

biodegradation of the PBAT samples. In the blank compost samples (Day 0), a complex

microbiota consisting of different bacterial species was present. Cloned gene sequences

representing members of bacterial phyla Proteobacteria, Bacteroidetes, Firmicutes,

Acidobacteria and others were present. Members of these phylogenetic groups are

commonly reported in mature composts (Dees and Ghiorse, 2001; Peters et al. 2000)

suggesting that the compost samples used in this study were representative of natural

microbiota present in composts worldwide. It was found that 46.6% of the clones were

most closely related to Phylum Proteobacteria, followed by members of Phylum

Bacteroidetes (Table 4.7).

After 100 days of composting, 50% of the clones in case of Blank compost (without any

polymer) and 40% of the clones in case of PBAT compost were found to be closely

related to Phylum Chloroflexi. The remaining phyla found in blank compost after day

100 were Bacteroidetes, Firmicutes, Acidobacteria, Actinobacteria, Proteobacteria and

Planctomycetes and the remaining phyla found in PBAT compost after day 100 were

Proteobacteria, Bacteroidetes, Firmicutes, Thermoflavimicrobium, Gemmatimonadetes,

Acidobacteria, Actinobacteria, Planctomycetes and unidentified bacterial clones.

The respirometric data (Table 4.6) suggested that biodegradation process had been

rapidly initiated in the PBAT composites and by day 100 almost 75% of it had been

mineralized.

68

Table 4.7 Microbial diversity in Day 0, Day 100 Blank and Day 100 PBAT composts

Phyla Class, subclass

or subdivision

Total number of

clones

Day 0

Total number of

clones

Day 100 Blank

Total number

of clones

Day 100

PBAT

Proteobacteria α - proteobacteria 8 2 3

Β - proteobacteria 1

γ - proteobacteria 2 2

δ - proteobacteria 4

Bacteroidetes Sphingobacteria 6

Flavobacteria 1

Unknown 4 2

Firmicutes Clostridia 1 3 2

Thermoflavimicrobium Unknown 1

Gemmatimonadetes Unknown 1

Acidobacteria Unknown 1 3 1

Verrucomicrobia Opitutae 1

Actinobacteria Micrococcineae 1

Unknown 3 2

Chloroflexi Unknown 1 15 12

Planctomycetes Unknown 1 1

Unidentified bacterial

clones

-- 4

= dominant bacterial Phyla in Day 0 compost,

= dominant bacterial Phyla in Day 100 Blank and Day 100 PBAT compost

69

Compost is the end-product of a biological decomposition and stabilization of organic

substrates under high temperatures as a result of biologically-produced heat. The

specificity and consistency of the bacterial communities inhabiting the compost

materials suggest that cultivation-independent bacterial community analysis is a

potentially useful indicator to characterize the microbial community and quality of

finished composts. It has been suggested that the microbial colonization in the finished

product is dependent on the following factors: a) the composition of the initial

substrates b) the processing/conditions that the compost is subjected to and c) the

quality of the finished substrate (Frachhia et.al. 2006). Specifically, compost pile

temperature and substrates available to bacteria appear to be the main factors

determining the bacterial community (Cahyani et al. 2003). Zhang et al. (2011) found

that bacterial community composition was significantly related to water-soluble carbon,

ammonium and nitrate during agricultural waste composting. Klamer and Bååth (1998)

studied microbial community dynamics during composting of straw and pig slurry and

found that there was a considerable difference with communities found during

composting of municipal solid waste, especially in the poor representation of

Actinomycetes species in the former.

70

An understanding of interactions between microorganisms and polyester composites is

useful for development of novel composites with desired properties for various

applications and developing sustainable infrastructure for their efficient waste disposal

after-use. However, because of the similarity in the abundance of the dominant phyla

(i.e. Chloroflexi), it seems the presence of PBAT composites have not triggered the

proliferation of any particular kind of bacterial microbiota. To the best of our

knowledge, this is the first in-depth report on microbial communities involved in the

biodegradation of chitin and PBAT composites. Members of Phylum Chloroflexi are

predominant members of activated sludge and are known degraders of cellulose. Their

involvement in biodegradation of synthetic polymer composites has not been studied or

indicated previously. An intensive study by Kragelund et al. (2008) found that

Chloroflexi constituted a specialized bacterial group, active under aerobic conditions

that primarily consume carbohydrates by secreting exo-enzymes, e.g. chitinase,

glucuronidase and galatosidase. Thermophilic members of these bacteria have also been

found with optimum temperature for growth around 55°C and optimum pH between 7

and 9. Chloroflexi have been found dominating in compost that had moisture profile

similar to the one examined here which ranged from 55-65% (Baharuddin et al. 2009,

Frachhia et al. 2006). They have been detected in various environments and are

generally hard to cultivate (Frachiaa et al. 2006). Indeed, it has been repeatedly

demonstrated that many not-yet-cultured microorganisms contribute to the composting

processes (Peters et al. 2000, Quaiser et al. 2003).

71

5. CONCLUSIONS AND FUTURE DIRECTIONS

The increased use of synthetically derived plastic packaging products and commodities

has led to the need for the development of environmentally friendly, biodegradable

plastics. MM Foods Pty Ltd., Melbourne has developed chitin-based biodegradable

composites for this purpose. The polymers contained either a large amount of

polyolefins or polyesters (PBAT/PBSA) in different combinations to aid in processing

and to provide the resulting materials with mechanical resistance. These polymers were

produced using a grinder, mixer, pelletiser and extruder. The constituents of each

composite were not subjected to any pre-processing chemical treatment whatsoever.

The purpose of this study was to assess the rate of biodegradation of these composites

according to the Australian Standard AS ISO 14855 (2005) and ascertain the effect of

the biodegradation on the bacterial community of the medium (compost in this case).

The mechanical testing of chitin composites showed typical behaviour attributable to a

non-compatible matrix-filler union. The tensile strength and elongation of pure PBSA

and PBAT decreased after combining with fillers. For all the composites (Fig. 3.2 and

Fig. 3.3), the tensile strength decreased markedly and continuously with increasing filler

content. This is attributable to the poor dispersion of chitin and MCC in the PBSA and

PBAT matrix. The presence of hydroxyl groups in the chitin and MCC fillers leads to

moisture uptake, resulting in poor interfacial bonding between fillers and the

hydrophobic matrices. The effect of this incompatibility on the mechanical properties

of the composites is substantial.

Future work will need to address these issues in order to produce hybrid composites

with desirable mechanical properties. Most surface treatments aim to compatibilise the

hydrophilicity of the fibre to the polymer to aid in polymer wetting, but some covalently

bond the two together or add surface roughness to the fibre to aid in mechanical

interlocking. Some of the chemicals and treatments that could be investigated for this

purpose include acids (hydrochloric acid, acetic acid), alkali (sodium hydroxide),

isocyanate treatments, silanation, reactions with acryl groups and esterifications using

anhydrides.

72

Investigation was undertaken to gauge the biodegradation rate of various chitin

composites. The very first composites which were tested by a simple soil-burial process

were chitin-LDPE composite films. After a period of one month it was observed that,

although weight loss in the chitin-LDPE surpassed that of neat LDPE, the difference

was not enough to consider the composite completely degradable. So, in keeping with

the aim of the study to develop completely biodegradable composites from chitin,

LDPE was partially replaced by Flex-262 (a cost-effective, commercially available

polymer which was found to be partly biodegradable in this study). Chitin-Flex262-

LDPE composites were subjected to biodegradation under controlled composting

conditions (in accordance with AS ISO 14855-2005). Respirometric data indicated that

the biodegradation was 22.6% after 6 months and therefore it can be concluded that the

presence of even partial amounts of LDPE hindered the biodegradation. This conclusion

was confirmed by FTIR analysis.

The logical next approach was to completely replace LDPE with biodegradable

polyesters. Flex-262 was also abandoned because of its resistance to undergo complete

mineralisation. Instead the focus was shifted to PBSA and PBAT which are

hydrocarbon-derived, readily available and certified 100% biodegradable polymers, to

be used as the materials of choice for building the polymer matrix. During composting

tests, the presence of both chitin and MCC had a strong influence on the biodegradation

of PBAT/PBSA composites which was monitored by CO2 emissions.

PBSA and PBAT composites made with chitin exhibited slower degradation rates

compared to neat PBSA and PBAT despite the former group showing weaker tensile

strength. It can be concluded that chitin as a biopolymer is somewhat more resistant to

microbial attack than neat PBSA and PBAT rendering the composites less readily

biodegradable under composting conditions. The resistance of chitin to bacterial attack

and its slow hydrolysis rate has been reported by others (Kirchman and White 1999;

Keyhani and Roseman 1999; McCarthy et al. 1997). The hydrolysis and biodegradation

rate of commercially available chitin has not been compared directly to those of readily

biodegradable polymers such as PBSA, PBAT in this study due to the limited number of

bioreactors that were available, however, the biodegradation rate of the composites

provide an indirect indication in support of the argument stated above.

73

The exception was the PBSAF10 composite which was found to behave equivalently to

its parent polymer PBSA in most tests. There was no significant difference between the

two in tensile strength as well as percentage mineralization in compost. On the other

hand there was observed a significant increase in modulus. The only difference was

observed in the reduced impact strength in case of PBSAF10. Considering the results

obtained PBSA with 10% chitin filler could be considered for future applications in

biodegradable plastic packaging.

In the next experiment, composite pellets made of chitin, MCC and PBSA/PBAT were,

however, found possess higher rate of degradation than neat PBSA/PBAT pellets (refer

to Table 4.6). In case of PBSA the difference was found to be significant (p value

0.016) when ultimate percentage mineralization was accounted for. The presence of

MCC, and the fact that native polyester pellets did not undergo extrusion and were used

as test materials for composting as received could have been factors in the results that

were observed. The degree of biodegradability of chitin-MCC-PBSA/PBAT composite

materials was similar to that of natural materials, such as cellulose. These materials

have a short survival time in biotic environments such as compost, and are therefore

suitable for disposal in landfills after their use.

The biodegradation process is a natural and complex phenomenon. Imitating natural

degradation processes is difficult to realise in the laboratory due to the great number of

parameters occurring during the biogeochemical activities. All these parameters cannot

be entirely reproduced and controlled in vitro. In particular, the effect of diversity and

efficiency of microbial communities (e.g. the complex interactions between microbial

communities and abiotic factors) and their abilities to use and to transform a variety of

nutrients is extremely difficult to anticipate. Nevertheless, biodegradability tests provide

necessary tools to estimate the environmental impact of industrial materials and to find

solutions to avoid waste disposal problems.

Molecular ecological techniques were used to identify a numerically dominant group of

potential PBAT composite-degrading bacteria belonging to the phylum Chloroflexi.

Members of this bacterial group were already detectable in the Day 0 compost and the

temperature and humidity conditions seem to have fostered their abundance as the

composting process progressed over time. There was no major discrepancy in the

microbial diversity or population of the blank compost and the PBAT composites after

74

100 days of composting which leads to the conclusion that incorporation of PBAT

composites in a 1:6 ratio to the compost had no effect on the normal development of the

bacterial community. Chloroflexi are abundant in the wastewater treatment processes

such as activated sludge processes and have been indicated in the degradation of

complex carbohydrates. Therefore, activated sludge processes and composting treatment

can be considered as one of the suitable waste management schemes for biodegradable

packaging waste made of polyesters like PBAT and PBSA and their composites.

A similar approach might be used to study biodegradation of other degradable

composites in different environmental systems such as soil, compost and seawater. An

understanding of the interactions between microorganisms and PBSA/PBAT

composites is useful for the development of novel composites with desired properties

for various applications and developing sustainable infrastructure for their efficient

waste disposal after-use.

75

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7. APPENDICES

1803F

Tensile stress at Break (Standard)

(MPa)

Tensile strain at Break (Standard)

(%)

Tensile strain at Maximum Load

(%)

Tensile stress at Maximum

Load (MPa)

1 18.69 145.37 20.48 24.18

2 18.15 15.74 16.77 22.45

3 18.52 10.09 10.07 18.80

4 17.87 103.15 22.90 22.72

5 18.74 52.75 22.05 24.35

Modulus (Automatic) (MPa)

Tensile stress at Yield (Zero Slope)

(MPa)

Tensile strain at Yield (Zero Slope)

(%)

Extension at Break

(Standard) (mm)

1 320.68 16.89 6.30 79.7310

2 304.84 17.44 6.59 47.3987

3 320.08 14.14 5.84 10.6480

4 297.62 15.73 7.10 96.4808

5 324.21 16.96 6.83 33.8133

1803F10

Tensile stress at Break (Standard)

(MPa)

Tensile strain at Break (Standard)

(%)

Tensile strain at Maximum Load

(%)

Tensile stress at Maximum

Load (MPa)

1 17.44 9.81 9.53 17.50

2 17.99 11.10 10.68 18.12

3 18.21 13.38 12.07 18.86

4 12.39 10.77 10.62 18.60

5 18.07 8.09 8.03 18.09

Modulus (Automatic) (MPa)

Tensile stress at Yield (Zero Slope)

(MPa)

Tensile strain at Yield (Zero Slope)

(%)

Extension at Break

(Standard) (mm)

1 491.08 13.31 4.13 8.9813

2 410.51 13.73 4.80 9.3978

3 480.09 14.02 3.96 11.9013

4 495.69 14.00 4.57 10.0679

5 533.63 14.12 4.06 7.8180

89

1803F20

Tensile stress at Break (Standard)

(MPa)

Tensile strain at Break (Standard)

(%)

Tensile strain at Maximum Load

(%)

Tensile stress at Maximum

Load (MPa)

1 14.16 4.46 4.19 14.50

2 14.97 5.22 5.17 15.19

3 14.91 4.50 4.50 14.91

4 15.15 4.52 4.43 15.23

5 14.73 4.99 4.50 15.12

Modulus (Automatic) (MPa)

Tensile stress at Yield (Zero Slope)

(MPa)

Tensile strain at Yield (Zero Slope)

(%)

Extension at Break

(Standard) (mm)

1 693.08 13.28 2.97 4.0698

2 759.53 13.50 3.10 5.2298

3 752.14 13.35 2.93 4.3982

4 826.94 13.33 2.71 4.5681

5 810.50 13.74 3.15 4.3998

2003F

Tensile stress at Break (Standard)

(MPa)

Tensile strain at Break (Standard)

(%)

Tensile strain at Maximum Load

(%)

Tensile stress at Maximum

Load (MPa)

1 11.66 339.33 339.19 11.66

2 10.04 282.95 278.66 10.61

3 8.80 203.96 200.87 9.54

4 9.77 257.18 254.23 10.71

5 8.57 233.82 231.61 10.43

Modulus (Automatic) (MPa)

Tensile stress at Yield (Zero Slope)

(MPa)

Tensile strain at Yield (Zero Slope)

(%)

Extension at Break

(Standard) (mm)

1 8.92 ----- ----- 303.3930

2 11.34 ----- ----- 257.4719

3 16.71 ----- ----- 194.6416

4 12.59 ----- ----- 234.7137

5 14.26 ----- ----- 222.0626

90

2003F10

Tensile stress at Break (Standard)

(MPa)

Tensile strain at Break (Standard)

(%)

Tensile strain at Maximum Load

(%)

Tensile stress at Maximum

Load (MPa)

1 8.37 188.59 187.18 8.55

2 5.68 218.46 214.97 8.69

3 7.88 218.39 214.76 8.07

4 8.73 235.91 232.95 8.88

5 8.31 244.83 238.12 8.55

Modulus (Automatic) (MPa)

Tensile stress at Yield (Zero Slope)

(MPa)

Tensile strain at Yield (Zero Slope)

(%)

Extension at Break

(Standard) (mm)

1 14.82 ----- ----- 174.5576

2 12.62 ----- ----- 192.6462

3 11.82 ----- ----- 183.2282

4 10.94 ----- ----- 209.9738

5 10.73 ----- ----- 202.2254

2003F20

Tensile stress at Break (Standard)

(MPa)

Tensile strain at Break (Standard)

(%)

Tensile strain at Maximum Load

(%)

Tensile stress at Maximum

Load (MPa)

1 6.18 13.73 10.70 6.63

2 5.59 12.85 9.05 5.90

3 6.18 14.39 9.45 6.50

4 5.91 17.79 9.95 6.27

5 6.14 13.62 10.00 6.46

Modulus (Automatic) (MPa)

Tensile stress at Yield (Zero Slope)

(MPa)

Tensile strain at Yield (Zero Slope)

(%)

Extension at Break

(Standard) (mm)

1 153.63 5.37 5.38 11.3146

2 157.26 4.81 4.55 10.2312

3 156.18 5.26 4.71 11.3146

4 153.81 5.08 4.46 13.2311

5 150.13 5.12 4.80 15.3175

91

PBSA-CHI/MCC

Tensile stress at Break (Standard)

(MPa)

Tensile strain at Break (Standard)

(%)

Tensile strain at Maximum Load

(%)

Tensile stress at Maximum

Load (MPa)

1 8.51 1.32 1.32 8.51

2 8.82 1.56 1.56 8.82

3 6.16 1.23 1.19 8.75

4 7.43 1.19 1.19 7.43

5 8.08 1.37 1.37 8.08

Modulus (Automatic) (MPa)

Tensile stress at Yield (Zero Slope)

(MPa)

Tensile strain at Yield (Zero Slope)

(%)

Extension at Break

(Standard) (mm)

1 1118.63 ----- ----- 1.4782

2 850.36 ----- ----- 1.6499

3 1062.80 ----- ----- 1.6499

4 916.71 ----- ----- 1.2299

5 1044.34 ----- ----- 1.3999

PBAT-CHI/MCC

Tensile stress at Break (Standard)

(MPa)

Tensile strain at Break (Standard)

(%)

Tensile strain at Maximum Load

(%)

Tensile stress at Maximum

Load (MPa)

1 5.79 7.77 7.17 5.85

2 5.65 11.40 9.56 5.80

3 5.70 7.33 7.02 5.79

4 5.80 10.24 8.24 5.95

5 5.37 10.17 8.38 5.57

Modulus (Automatic) (MPa)

Tensile stress at Yield (Zero Slope)

(MPa)

Tensile strain at Yield (Zero Slope)

(%)

Extension at Break

(Standard) (mm)

1 183.10 4.71 3.43 7.5681

2 165.63 4.58 3.88 9.2346

3 188.90 4.51 3.49 6.9814

4 193.51 4.64 3.36 9.3978

5 175.47 4.48 3.61 9.2328