“assessment of biodegradation in different environmental ... · “assessment of biodegradation...

Research Doctorate School in Biological and Molecular Sciences

Ph D. in BIOMATERIALS – XXV Cycle (2010-2012)

“Assessment of Biodegradation in Different

Environmental Compartments of Blends and

Composites Based on Microbial

Poly(hydroxyalkanoate)s”

Hayan Chen

Supervisor: Prof. Emo Chiellini Co.Supervisor: Dr. Brunello Ceccanti*

Tutors: Dr. Lucia Pérez Amaro, Dr. Arianna Barghini, Dr. Andrea Corti

Dr. Serena Doni*, Dr. Grazia Masciandaro*

Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental

Applications (BIOlab) – Department of Chemistry and Industrial Chemistry

(University of Pisa)

*National Research Council (CNR)-Institute of Ecosystem Study (ISE)

Index

INDEX ACKNOWLEDGMENTS ...................................................................................................... 1

ABSTRACT ............................................................................................................................ 3

1. INTRODUCTION .............................................................................................................. 4

1.1. Global And European Production/Consumption of Plastic And Polymers

Materials ................................................................................................................... 4

1.2. The Advantages And Disadvantages of Conventional Packaging Materials ......... 6

1.3. The Probably Solutions Based on the Available Investigations .............................. 7

1.3.1 Waste Materials Disposal and Potential Risks ................................................... 10

1.4 Development of Polymeric Materials from Renewable and Fossil Fuel Resources,

Biopolymers and Relevant Plastic Items. ............................................................. 11

1.4.1. Poly (hydroxyalkanoates) (PHAs) ..................................................................... 12

1.4.2. Poly (lactic acid) (PLA) .................................................................................... 15

1.5. Different Media as Tool Boxes for Biodegradation of Plastic .............................. 16

1.5.1. PHAs Biodegradation in Soil ........................................................................... 17

1.5.2. PHAs Biodegradation in Mature Compost ........................................................ 21

1.5.3. PHAs Biodegradation in Aqueous Medium ....................................................... 23

1.5.4. Microbial Biodegradation Mechanism (intra and extra-cellular) ..................... 27

1.5.5. PHAs Biodegradation Provided by Enzymes .................................................... 29

1.5.6. PLA Biodegradation Mechanism in Compost ................................................... 31

1.5.7. The Role of the Organic Filler on the Biodegradation Behavior and Final

Properties of PHAs Composites. ...................................................................... 34

1.5.7.1. PHAs-Cellulose Derivatives Blends and Composites ...................................... 35

1.5.7.2. PHA-Natural Fiber Composites ....................................................................... 36

1.5.7.3. PHAs Lignin Blends and Composites .............................................................. 40

1.5.8 The Role of the Organic Filler on the Biodegradation Behavior and Final

Properties of Petro-based Polymers Composites (PE, PP, PS) ........................ 41

1.5.9. The Role of the Inorganic Filler on the Biodegradation Behavior and Final

Properties of PHAs .......................................................................................... 45


Properties of Petro-based Polymers (PE, PP, PS) ........................................... 50

1.6 Biodegradation of Fossil Fuel Based Polymers (ECOFLEX, PE and PS) in Soil-

Plant Systems (Avena sativa –Raphanus sativus) ................................................ 53

1.6.1. Biodegradation of plastics in soil ...................................................................... 55

1.6.2. Polyethylene ...................................................................................................... 56

1.6.3. Polystyrene ........................................................................................................ 58

1.6.4. Aliphatic–aromatic (AAC) copolyesters ............................................................ 58

1.7 Polyvinylics Biodegradation ..................................................................................... 59

1.7.1 The Role of Plastics in Agriculture ..................................................................... 60

1.7.2 Biodegradation of pro-oxidant-free polyethylene by natural occurring

microorganisms ................................................................................................ 63

1.7.3 Preliminary data or studies ................................................................................ 65

Chen Haiyan PhD Thesis

2. OBJECTIVES .................................................................................................................. 73

3. EXPERIMENTAL PART ............................................................................................... 75

3.1. Materials .................................................................................................................. 75

3.1.1. Reagents and Solvents ...................................................................................... 75

3.1.2. Joncryl ADR-4368-CS ...................................................................................... 75

3.1.3. Hazelnut Shells ................................................................................................. 76

3.1.3.1. Lignin Functionalization ................................................................................. 77

3.1.4. Organophilic Clay (Dellite 72T) ...................................................................... 77

3.1.5. Perkalite F100 .................................................................................................. 78

3.1.6. Poly(3 hydroxy-co-3 valerate) (PHBV) ............................................................ 78

3.1.7. Poly(lactic acid) (PLA) ..................................................................................... 81

3.1.8. Poly(butylene adipate-co-terephthalate) (Ecoflex)........................................... 82

3.1.9. Ecovio ............................................................................................................... 83

3.2. Preparation of the Sample ...................................................................................... 84

3.2.1. Pre-blend .......................................................................................................... 84

3.2.2. Melt blending .................................................................................................... 84

3.2.3. Press ................................................................................................................. 85

3.3 Characterization of the Samples ............................................................................. 86

3.3.1. Thermogravimetric Analysis (TGA) ................................................................. 86

3.3.2. Differential Scanning Calorimetry (DSC) ........................................................ 86

3.3.3. Transmision Fourier Transform Infrared Spectroscopy (FTIR) ....................... 86

3.3.4. Nuclear Magnetic Resonance (1H-NMR) ......................................................... 86

3.3.5. Gel Permeation Chromatography (GPC) ......................................................... 86

3.3.6. Determination of Total Organic Carbon (TOC) and Total Nitrogen (TN) ....... 87

3.3.7. PH and Electrical Conductivity ........................................................................ 87

3.4. Experimental Setup for Biodegradation Experiments ......................................... 87

3.4.1. Soil Burial Biodegradation Tests...................................................................... 87

3.4.2. Mature Compost Respirometric tests ................................................................ 91

3.4.3. River Water respirometric Test ........................................................................ 93

3.4.4. Carbon Dioxide Determination and Evaluation of the Degree of

Biodegradation in Respirometric Tests ........................................................... 94

3.5. Specific Biotests to Assess Eco-toxicity of Biodegradable Polymer Materials

Ecoflex, PE, PS in Soil-Plant systems .................................................................. 95

3.5.1 Materials ........................................................................................................... 95

3.5.1.1. Soil and Plants used in the experiments .......................................................... 95

3.5.1.2. Testing materials ............................................................................................. 96

3.5.2. Experimental Setup ........................................................................................... 97

3.5.2.1. Degradation in a soil-plant system .................................................................. 97

3.5.2.2. Biodegradation of PHB in soil ........................................................................ 97

3.5.3. Methods ............................................................................................................ 97

3.5.3.1. Chemical Parameters (pH, electrical conductivity, total organic carbon, total

nitrogen, water soluble carbon, nitrate, ammonia, total and available

elements) ....................................................................................................... 97

3.5.3.2. Biochemical Parameters ................................................................................. 98

Index

3.5.3.3. Biological parameters ...................................................................................... 99

3.5.3.4. Germination and Growth Test ......................................................................... 99

3.5.4. Statistical Analisys ............................................................................................ 99

4. RESULTS AND DISCUSSION ..................................................................................... 101

4.1. Differential Scanning Calorimetry (DSC) ............................................................ 101

4.1.1. PHBV Based Blends and Composites.............................................................. 101

4.1.2. PLA Based Blends & Composites ................................................................... 106

4.2 ThermoGravimetric Analysis (TGA) .................................................................... 110

4.2.1. PHBV Based Blends & Composites ................................................................ 110

4.2.2. PLA Based Blends & Composites ................................................................... 114

4.3. Molecular Weight by Gel Permeation Cromatography (GPC) Analysis .......... 118

4.4 PHAs Based Composites Processing Characteristic ............................................ 119

4.5. PHBV Based Blends and Composites Structural Characteristic by Infrared

Spectroscopy (IR) ................................................................................................. 127

4.6. Biodegradation Experiments ................................................................................. 129

4.6.1 PHBV Based Blends and Composites in Soil Medium ..................................... 130

4.6.2. PHAs Based Blends and Composites Biodegradation in Mature Compost ..... 132

4.6.3. PLA Based Blends and Composites Biodegradation in Mature Compost ....... 133

4.6.4 Qualitative Compost Experiment ..................................................................... 134

4 .6.5. River Water Respirometric Test ..................................................................... 136

4.7. Biodegradation of Fossil Fuel Based Polymers (ECOFLEX, PE and PS) in Soil-

Plant Systems (Avena sativa –Raphanus sativus) ................................................ 138

4.7.1. Potential Eco-toxicological Effects of Polymer Materials .............................. 138

4.7.2. Soil biochemical parameters .......................................................................... 139

4.7.3. Chemical parameters of soil samples treated with different polymers ............ 142

4.7.4. Statistics analyzes about Biplot PC1 versus PC2 ............................................ 144

4.8. PHB biodegradation in soil ................................................................................... 149

5. CONCLUSIONS ............................................................................................................. 151

REFERENCES ................................................................................................................... 154

Acknowledgment

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ACKNOWLEDGMENTS

It is pleasure to thank everybody who made this thesis possible including my supervisory

team, my family. I am very thankful to financial support by Research Doctorate School in

Biological and molecular Sciences, Laboratory of Bioactive Polymeric Materials for

Biomedical and Environmental Applications (Biolab), Department of Chemistry, University

of Pisa, Italy (UNIPI) and the National Research Council (CNR)-Institute of Ecosystem

Study (ISE). Part of this work was performed within framework of EC-funded project:

Animpol CT: GA-245084, the financial support is greatly acknowledged.

First of all, I would like to express my deepest sense of gratitude to my principal supervisor,

Prof. Emo Chiellini, who gave me the opportunity to achieve this important objective, his

able guidance and constructive critism helped me to progress in research pursuit, I am

thankful for his constant encouragement and valuable contribution of his time throughout the

course of my thesis; and sincere thanks to my secondary supervisor, Prof. Brunello Ceccanti,

who trust me and quickly discovered my potential and interest in the research area, it‘s been

a great honor for me to work under his inspiring guidance; my gratitude goes to Prof. Yao

Jun, my previous supervisor during my master degree, for her constant encouragement and

guidance.

My sincere thanks to my tutor Dr. Andrea Corti, who is sharing his academic experience for

the successful completion of the objective undertaken. His valuable suggestions and timely

discussions enabled me to achive this work with precision. Secondly, I would like to express

my heartfelt gratitude to Dr. Serena Doni, Dr. Lucía Pérez Amaro and Dr. Arianna Barghini,

who led me into the world of scientific methodology, the standard training of chemical,

engineering, biological experimental epistemology and technical ability, they has walked me

through all the stages of the writing of this thesis. Without their consistent and illuminating

instruction, this thesis could not have reached its present form.

I also very thank the reading committee members who kindly agreed to read my manuscript

and participate in this discussion. And many thankful to Prof. Mingreng Wang, professorate

senior engineer, China GuoDian（Group）Corporation, for his suggestions and discussions

during the part of my research work.

I am very much thankful to Dr. Federica Chiellini, Dr. Andrea Morelli, Dr. Vassilka IIieva, Dr.

Stefania Cometa, and Dr. Marcella Ferri during laboratory work.

My special thanks to Maria Caccamo, Maria Viola, and Elisa Romano. Persons that help me

in all situations!

I would like to thank several people from the BIOLAB for all the time we spent together in

these years, for share the good times and for the help that they gave me in the mo ments I

needed. Great colleagues like, Anna Maria Piras, Antonella Lisella, Alberto Dessy,

Alessandro Pirosa, Carlos Mota, Cesare Errico, Chiara Migone, Cristina Bartoli, Chongbo

Chen, Dino Dinucci, Dario Puppi, Dash Mamoni, Giulia Ciampi, Francesca Scalesse,

Muniyasamy Sudhakar, Matteo Gazzarri, Mohamed Abdelwahab, Stefania Sandreschi, Silvia

Volpi, Kaili Yang, Xuanmiao Zhang and all the others that are too long to list.

I also would like to thank several people from the ISE-CNR for all the time we spent

together in these years, for share the good times and for the help that they gave me in the


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moments I needed. Great colleagues like, Cristina Macci, Eleonora Peruzzi, Grazia

Masciandaro, Giulia Bondi, Mariarita Arenella, and all the others people helped me during

three years.

Finally, my thanks greatly indebted go to my beloved family for their loving considerations

and great confidence in me all through these years, they always encouraged me in all

moments providing emotional support and love for my carrier. I also owe my sincere

gratitude to my good friends and my officemates, fellow classmates who gave me their help

and time in listening to me and helping me work out my problems during the difficult course

of the thesis.

Introduction

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ABSTRACT

The packaging industry nowadays relies strongly on the use of petroleum-derived plastic

materials, which raises some concerns from both economical and environmental perspectives.

The continuing use of finite oil resources implies a likely eventual decrease in availability

and thereby increasing costs of raw materials. Furthermore, being non readily biodegradable,

petroleum-based products can pose significant waste disposal problem in some area.

Packaging is a substantial part of our everyday life, and the consumer use of packaging

materials has shown a continuous increase over time. Packaging products manufactured from

renewable materials currently only represent about 2% of the market, except the traditional

fiber-based packaging.

The research work of my doctoral thesis, performed in the BIOLAB (Laboratory of

Bioactive Polymeric Materials for Biomedical & Environmental Applications, Department of

Chemistry and Industrial Chemistry, University of Pisa) facilities deals with the assessment

of the biodegradation propensity in different media (soil, compost, river water) of synthetic

polyesters (Ecoflex and Ecovio) and bio-based polymers such as poly(hydroxybutyrate-co-

hydroxyvalerate) (PHBV), poly(lactic acid) (PLA) and their blends and composites loaded

with fillers of organic (lignin), inorganic (montmorillonite named Dellite) nature and chain

extenders (Joncryl).

The composites obtained by melt-blending and the neat polymers have been analyzed also

for their processing profile and thermal properties (TGA, DSC) with the aim to know the

effect of each component on the overall composite biodegradation behavior.

The experiments performed at the CNR-ISE (National Research Council-Institute of

Ecosystem Study) have been essentially two topics.

In the first investigation, the potential eco-toxicological effects of polymer materials such as

Polyethylene (PE) and Polystyrene (PS) in soil-plant systems (Avena sativa and Raphanus

sativus) were studied; the PE and PS polymer effects were compared with those of the

biodegradable materials Ecoflex and Cellulose, used as positive controls. Several chemical

and biochemical parameters, usually used to assess soil quality and its response to xenobiotic

compounds, have been selected in order to follow the effect of biopolymer degradation on

soil properties. Polymers seemed to stimulate the soil metabolic activity during the time, thus

indicating their possible biodegradation propensity in soil. These effects were more evident

in Raphanus sativus plant treatments than in Avena sativa, suggesting the higher sensitivity

of the former to the polymer treatments. At the end of experimentation, Ecoflex compared to

PE and PS showed the higher nitrate/ammonia ratio and the lower water soluble carbon

content (WSC), thus indicating its higher ability to promote the carbon cycle and in turn to

activate organic nitrogen metabolism, bringing the soil plant system towards the nitrate

nutrient production.

In the second experiment, the biodegradation of the PHB polymer, added to the soil at two

different concentrations (1% and 2%) was investigated. The biodegradation process was

followed by monitoring specific microorganisms and enzyme activities involved in PHB

metabolism. Total microbial cultivable population and Pseudomonas genus significantly

increased in both PHB dose treatments with respect to the untreated soil. The stimulation of

microbial growth and activity was confirmed by the activation of the arylesterase and PHB

depolymerase activities.


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1. INTRODUCTION

1.1. Global and European Production/Consumption of Plastic and Polymers Materials

Synthetic and bio-based plastics technology was developed not only as expected to continue

its unique properties for more and more innovative applications, The recalcitrance of some

plastic items to biodegradation has led to an increase public concern about the hazards

associated with plastic residues in the environment has triggered activities by scientific,

industrial, and regulatory bodies to assess the exposure and effects of these macromolecular

compounds in different environmental compartments during past decades

(www.plasticeurope.com). The synthetic plastics are definitely important in modern daily life

and have brought many societal benefits. They show some advantages such as low price,

easy disposal, physical, chemical and thermo-mechanical properties thermo property,

physical and chemical stability, that make them very convenient materials for industrial

process and consumption. A wide variety of synthetic polymers are produced worldwide to

the extent of approximately 300 million tons per year and remarkable amounts of these

polymers are introduced in the ecological system as industrial waste products (Shah et al.

2008).

Global plastics production increased by 10 million tonnes (3.7%) to around 280 million

tonnes in 2011 (Fig.1.1), continuing the growth pattern that the industry has enjoyed since

1950 approximately by 9% per annum (www.plasticeurope.com).

Figure 1.1. Global Plastic Production Until 2011.

Figure 1.2 reports the European plastic demand by resin type, whereas Figure 1.3 shows the

demand segmentation by sector for the year 2011.

http://www.plasticeurope.com/

Introduction

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Figure 1.2. European Plastic Demand by Resin Type 2011 (Year 2011).

Figure 1.3. European Plastic Demand by Sector (Year 2011).

The biobased world production raises 1161 k-tons in the year 2011 as observed from Figures

1.4 and 1.5. The last one reports the capacity for the biodegradable biobased materials and

the non-biodegradable ones in 2011 (European Bioplastics Bulletin, Issue 05/2012).


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Figure 1.4. Biobased Plastics World Production.

(a) (b)

Figure 1.5. Bioplastics Production Capacity or Biobased Biodegradable Materials (a) and

Biobased non Biodegradable Ones (b).

1.2. The Advantages and Disadvantages of Conventional Packaging Materials

Polyolefins are widely used, and it was reported that they accumulate in the environment at a

rate of 25 million tons per year (Orhan et al. 2000, Eubeler et al. 2010). The thermoplastic

such as polyethylene (PE) and polystyrene (PS) have a broad scale of applications in the past

few years. Whist polyethylene is available in many varieties (liner low density, high density,

high molecular weight, etc.), the low-density polyethylene (LDPE) is used extensively in

rigid packaging industry because of several favorable physical properties, for instance its low

vicat softening point aids in low temperature forming and heat sealing, which makes it very

favorable in the form-fill and seal-packaging industry (Paabo et al. 1987, Dilara et al. 2000),

and it attributes the form-fill and seal package industry to optimize equipment cycle-times.

Low density polyethylene (LDPE) shows general properties such as low cost, chemical

resistance, food grades available, good moisture barrier properties that make it one of most

life-partner materials in our daily life. In fact, LDPE is often used in conjunction with

another polymer like polystyrene (PS), for the improvement of the moisture barrier

transmission properties. The faeatures (optical clarity and thermal stability, easy processing

Introduction

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conditions, good property retention and creep resistance, toughness) similar to polyethylene,

make it suitable mainly for packaging applications (Lim et al. 1999, Arkedi et al. 2007).

The literature research considers also other application field such as the production and use

of the agricultural plastic films (Mulching by putting a thin plastic film directly over the soil

surface) as a standard technique (green house) for vegetable.

The advantages and purposes by using these films are (1) the control and/or the increase of

the soil temperature, (2) the maintaining of soil humidity, (3) the reduction of germination

time, providing early or out of season crop production for better market value products, (4)

the reduction for weeds and plant diseases, guarantying the efficiency in the usage of water

and fertilizers with the aim to improve the quality of horticultural products (Lamont 1999,

Sedlacek et al .2011, Espì et al. 2006, Kijchavengkul, et al. 2008)

On the other hand, the dramatic increase in production and lack of biodegradability of

commercial polymers, particularly commodity plastics used in packaging, industry and

agriculture, focused public attention on a potentially huge environmental accumulation and

pollution problem that could persist for centuries. About 40% of such production is discarded

into landfills (Shah et al. 2008). The plastic waste is disposed off through landfilling,

incineration and recycling. Because of their persistence in our environment, several

communities are now more sensitive to the impact of discarded plastic on the environment

(Shah et al. 2008, Albertsson et al. 1987). Despite all advantages of these conventional

polymers, there are several major concerns about using films which are the costs of removal

and disposal of the used plastics and environmental issues..

1.3. The Probably Solutions Based on the Available Investigations

The American Society for Testing and Materials (ASTM) and the International Organization

for Standardization (ISO) give some definitions: a) degradation as ―An irreversible process

leading to a significant change for the material structure, typically characterized by a loss of

properties (e.g. integrity, molecular weight, structure and mechanical strength) and/or

fragmentation (Figure 1.6) (Chiellini 2007). Degradation is affected by environmental

conditions and proceeds over a period of time comprising one or more steps‖ (ISO-472,

ASTM 883-1991, ASTM D6400-04).

Figure 1.6 Polymeric Materials and Plastics Nomenclature


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Biodegradable plastic is ―A degradable plastic in which the degradation results from the

action occurring by microorganisms such as bacteria, fungi and algae‖. Two main types of

commercially viable biodegradable plastics have been developed:

1) Oxo-biodegradable polymers and hence relevant plastic items, for which degradation is

the result of oxidative and cell mediated phenomena, either simultaneously or step by step;

2) Hydro-biodegradable polymers and hence relevant plastic items, for which degradation is

the result of hydrolytic and cell-mediated phenomena, either simultaneously or step by step.

For both types of biodegradable polymers, the molar mass reduction in the environment,

follows two sequential steps, where the first one is basically an abiotic degradation (Figure

1.7) whereas the second one represents a bioassimilation of the molecular fragments that are

generated in the first stage (Scott 1999). Abiotic mechanisms are generally regarded as too

slow by themselves to be adequate in a variety of disposal environments.

Figure 1.7. General Features of Environmentally Degradable Polymeric Materials.

There are several applications in which really quite rapid degradation of plastics after use is

required. For example, there is a lot of plastics disposable by chemical, physical or biological

treatments in liquid or sewage sludge system. The non-toxic products can be reused or

recycled in the environment as fertilizer, in which situation they lose their integrity

relatively, in order to avoid plugging pumps, filters and so on. Hydrolytically unstable

biodegradable plastics can provide an answer here. In many other uses (e.g., food

packaging), one of the disadvantages is hydrolytic instability. However, overall stability is

required during shelf storage and use, even though this method should follow relatively rapid

abiotic degradation within a specific time, which probably is depending on the disposal

environment. In order to avoid the accumulation of plastic fragments in virtually all disposal

environments, it requires that these be consumed through biodegradation by microorganisms.

Introduction

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Effective biodegradation of such residues can be achieved when originally hydrophobic

plastics become wettable (hydrophilic) and characterized by a relatively low molecular

weight so that there is a significant number of molecular ends accessible at the surface. The

science and technology of the development of oxo-biodegradable plastics can meet these

criteria (Muniyasamy 2011).

The mechanisms of oxidative degradation of polymers, have been extensively studied and

reviewed (Chiellini et al. 2003, Scott 1965). It is generally accepted that the key

intermediates are hydro-peroxides, which decompose under the influence of heat, light or

transition metal catalysis, producing free radicals. They enter a chain reaction with oxygen

and C-H bonds (hydrocarbonyl) in the polymer, to produce a range of oxidation products.

This can be expressed as the interlocking cycle of reactions depicted in Figure 1.8.

Figure 1.8. Biodegradation Routes for Oxo- and Hydro-Biodegradable Polymers.

According to the earlier research, a number of polymeric materials has been developed with

varying degrees of success (Rutiaga et al .2005, Mecking 2004, Chandra et al. 1998).

The biodegradable mulching films represents a really promising alternative to the common

plastic films (mostly polyethylene and polystyrene) for enhancing sustainable and

environmentally friendly agricultural activities. These biodegradable mulching films can be

incorporated into the soil at the end of the crop season and undergo biodegradation by soil

microorganisms. The possible biodegradation of the plastic mulches can abate several major

concerns about their wide usages: (1) costs of removal and disposal (2) environmental issues

(3) possible toxic effects on plant germination and growth. Only extensive analysis over the

entire life cycle of a particular polymer application can assess eventual risks for the

environment and plant health and, a selection of safer raw material can be made.

Additionally, some additives such as pro-oxidants and starch have been applied in science

materials, with the aim to improve the biodegradation propensity of the materials.


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Recent researches have showed that thermoplastics derived from polyolefins, traditionally

considered resistant to biodegradation in the environment, can biodegrade following a photo-

degradation or a chemical degradation mechanism (Zheng et al. 2005). Thermoset plastics,

such as aliphatic polyesters and polyurethanes, can be attacked by microorganisms that break

down the hydrolytic bonds contained in the esters or urethanes chemical structure.

Aliphatic-co-aromatic polyesters, such as Ecoflex and Ecovio, have e commercial

applications because they show good mechanical properties and biodegradation propensity.

(Yamamoto et al. 2002, Muller et al. 2001).

Ecoflex has been reported to biodegrade easily under compost conditions (Yamamoto et al.

2005). At high temperature, the material reached a complete degradation in 22 days of

incubation without showing any adverse eco-toxicological effects on the environment (Witt

et al. 2001). This lack of toxicity represents an evidence that Ecoflex could be an excellent

material for the production of short-life products such as protective packaging and films for

agricultural applications. The analyses were carried out under thermophilic conditions to

reproduce those in municipal or industrial composting facilities. The biodegradation

experiments were also performed under moderate environmental soil conditions with 29

strains of enzyme-producing soil bacteria, fungi and yeasts. A screening procedure was

developed and the results showed that, after 21 days of exposure, the polymer could be

degraded by some of the microorganisms (MOs) (Eubeler et al. 2010).

Degradation of poly [(1, 4-butylene terephthalate)-co-(1, 4-butylene adipate)] (Ecoflex,

BTA) monofilaments (rods) in standardized sandy soil was investigated (Rychter et al.

2010). Changes in the microstructure and chemical composition distribution of the degraded

BTA samples were evaluated and changes in the pH and salinity of post-degradation soil, as

well as the soil phytotoxicity impact of the degradation products, are reported.

A macroscopic and microscopic evaluation of BTA surface of some rod samples after

specified periods of incubation in standardized soil, indicated that its erosion starts from the

fourth month of their incubation, with almost total disintegration of the incubated BTA

material observed after 22 months. However, the weight loss after this period of time was

about 50% and only a minor change in the Mw of the investigated BTA samples was

observed, along with a slight increase in the dispersity (from an initial 2.75 up to 4.00 after

22 months of sample incubation). The multidetector SEC and ESI-MS analysis indicated

retention of aromatic chain fragments in the low molar mass fraction of the incubated

sample. Phytotoxicity studies revealed no visible damage, such as necrosis and chlorosis, or

other inhibitory effects, in the following plants: radish, cress, and monocotyledonous oat,

indicating that the degradation products of the investigated BTA copolyester are harmless to

the tested plants (Rychter et al. 2010).

1.3.1 Waste Materials Disposal and Potential Risks

Many of the wastes produced today remain in the environment for hundreds years so the

creation of non-decaying waste materials, combined with a growing consumer population,

could produce a waste disposal crisis. One solution to solve this crisis is constituted by the

solid waste management (Arnio et al. 2008) that provides some rules with the aim to recycle

waste into useful products.

For this purpose, different waste management techniques such as material recovery facilities,

landfilling, a source separation system able to distinguish the recyclable materials, a system

Introduction

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suitable for the recovery of the organic fraction, an incineration process and a composting

facility were investigated only on an environmental point of view with the aim to obatain

decision-making tools that consider the economic and social effects of solid waste

management (Banar et al. 2009, Huang 1995).

A large range of materials such as metal, glass, wood, paper or pulp-based materials, plastics

or combination of more than one materials as composites are used for packaging

applications. Among other materials, a huge range of oil-based polymers is also currently in

use. They are largely non-biodegradable so it is difficult to recycle or to reuse them due to

the levels of contamination (Davis et al. 2006).

Implementation of more fundamental regulatory testing procedures which reflect the

behavior of materials in end use applications, is recommended to allow better control over

environmental impact in the wide variety of utilization and disposal practices (Van der Sloot

1990).

Another option adopted to reduce the waste disposal is the development and production of

biodegradable materials with similar functionality to that of the oil-based synthetic polymers.

It is anticipated that, as the materials are from renewable resources and biodegradable, they

would contribute to sustainable development and if properly managed would reduce their

environmental impact upon disposal methods (Davis et al. 2006, Van der Sloot 1990,

Consonni et al. 2011, Dias et al. 2007).

The goal of this sustainable growth should be a reaching of a maximum level for the

efficiency of energy utilization in every step of the system, from the production of the goods

to the disposal of the wastes. Many highway agencies, private organizations, and individuals

have completed or are in the process of completing a wide variety of studies and research

projects concerning feasibility, environmental suitability, and performance in using recycled

products in highway construction. (Subramanian 2000). These studies try to meet society's

need for safe and economical disposal of waste materials with the highway industry's need

for better and more cost-effective construction materials.

Bio-based packaging material is defined as a packaging containing raw materials derived

from agricultural sources such as starch and/or man-made monomers. These materials are not

necessarily biodegradable (Petersen et al. 1999, Goddard 1995). Some articles and current

researchers are focused on those waste materials that become promising as substitutes for

conventional materials (Petersen et al. 1999, Goddard 1995).

Environmental issues are becoming increasingly important to the European consumer.

Consequently, consumer pressure may attract the attention of research and usage on bio-

based packaging materials as an alternative to materials produced from resources that don‘t

have renewable ability (Goddard 1995).

1.4 Development of Polymeric Materials from Renewable and Fossil Fuel Resources,

Biopolymers and Relevant Plastic Items.

Natural polymers and most synthetic macromolecules cannot be assimilated by

microorganisms. Although polymers represent slightly over 10% of total municipal waste,

the problem of non-biodegradability is highlighted by overflowing landfills, polluted marine

waters, and unsightly litter so the existing government regulations in Europe and anticipated


- 12 -

regulations in the United States will greatly limit the use of these materials in large volume

applications (packaging, water treatment, paper and textile sizing, etc.) unless acceptable

means of waste management are available.

Total management of polymer wastes requires complementary combinations of

biodegradation, incineration, and recycling. Biodegradation is the most desirable long-term

future solution and requires intensive research and development before it becomes practical.

On the other hand, incineration and recycling can become operational in a relatively short

time for the improvement of the situation at present and in the near future (Hoang et al.,

1995). Waste disposal is an increasing problem as available landfill areas diminish. Plastics

currently account for about 7% by weight (18% by volume) of municipal solid waste, with

half of this plastic waste derived from plastics used in packaging. Growing environmental

concern has created an urgent need to develop new biodegradable materials that have

properties comparable with conventional polymeric materials such as polyethylene and

polypropylene. To reduce the environmental impact of synthetic plastics, some industries

have started to produce biodegradable plastics (Rosa et al. 2003).

Suitable alternative methods such as biodegradable sodium alginate-based spray mulch,

biobased polymers films, are used in agriculture (De Prisco et al. 2002, Immirzi et al. 2009,

Malinconico et al. 2002, Dincheva et al. 2007). At the end of their life, biodegradable

materials can be integrated directly into the soil where bacterial floras transform them into

carbon dioxide or methane, water, and biomass. So they could represent a sustainable

ecological alternative to LDPE films (Immirzi et al. 2009). Among the wide range of

biopolymers available, polysaccharides are very interesting materials for film applications

because they are widely available and are renewable (Malinconico et al. 2002, Avella et al.

2007).

The innovative approach consisted of forming the mulch coating directly in field by covering

the soil with a thin protective geo-membrane obtained by spraying water-based solutions of a

natural polysaccharide, such as sodium alginate (NaAlg), glucomannan, chitosan and

cellulose.

Barrier coatings were obtained when the water content of polymeric network was removed

through evaporation. The spray technology used was similar to that used to apply fertilizers,

pesticides, hormones and other useful plant health products.

Nowadays, the adoption of a new material becomes a need for the manufacturing industry

that has to well know its behavior under various processing, manufacturing and usage

conditions (Sen et al. 2009). Various blends of conventional and biodegradable plastics have

been studied (Guo et al. 1999). Polymeric blends consist of a mixture of two or more

polymers or copolymers and they are a good alternative for reducing the final product cost.

Natural additives such as starch, cellulose, lignin, and other polysaccharides have been used

in these formulations (Tseng et al., 2007).

1.4.1. Poly (hydroxyalkanoates) (PHAs)

Poly(hydroxyalkanoate)s (PHAs) are a class of biopolyesters mainly produced by microbial

fermentation of renewable fossil fuel feedstocks or combination of both types of sources

(Reddy et al. 2003).

Introduction

- 13 -

The PHAs accumulate as granules within the cytoplasm of cells and serve as a microbial

energy reserve material (ota, 1993).

PHAs have a semicrystalline structure, the crystallinity degree ranging from about 40% to

around 80% (Abe & Doi, 1999).

PHAs have a generic formula where x is 1 and R can be an alkyl chain (C1-C15) (Fig. 1 &

Tab. 1.1).

H COO (CH2)x OH

n

CH

R

Figure 1.9 Generic formula structure of PHAs

The main members of the PHA family are:

poly(3-hydroxybutyrate), P(3HB), [R= CH3]

poly(3-hydroxyvalerate), P(3HV), [R= ethyl]

poly(3-hydroxyhexanoate), P(3HHx), [R= propyl]

poly (3-hydroxyoctanoate), P(3HO), [R= pentyl]

poly(3-hydroxydecanoate), P(3HD), [R= heptyl]

the medium-chain-length poly(3Hpd), [R= pentadecyl].

PHAs copolymers vary in the type and proportion of monomers, and they are typically

random in sequence. All the reported monomers and relevant monomeric units are

characterized by the presence of an asymmetric carbon atom characterized by (s)

configuration (Chen et al. 2001).

Table 1.1 shows some PHA molecular structures.

Table 1.1. Structures of Polyhydroxyalkanoates (PHAs).

PHA short name PHA full name χ R

PHB P(3HB) 1 -CH3

PHV P(3HV) 1 -CH2CH3

PHBV P(3HB-co-3HV) 1 -CH3 and -CH2CH3

PHBHx P(3HB-co-3HHx) 1 -CH3 and –CH2CH2CH3

PHBO P(3HB-co-3HO) 1 -CH3 and –(CH2)4CH3

PHBD P(3HB-co-3HD) 1 -CH3 and –C6H8CH3

PHBO P(3HB-co-3HOd) 1 -CH3 and –(CH2)14CH3

P(HB4HB) P(3HB-co-4HB) 2 -CH3 and –H

P(HB4HV) P(3HB-co-4HV) 2 -CH3 and –CH3

The range of PHA structural architectures that is now accessible has opened up a wide range

of possibility, encompassing rigid thermoplastics, thermoplastic elastomers, as well as

suitable grades for waxes, adhesives, and binders (Metabolix, 2007a, Ishida et al. 2005).

Feedstocks currently being utilized for PHA production are high value substrates such as

sucrose, vegetable oils and fatty acids (Li et al. 2007, Suriyamongkol et al. 2007). In theory,


- 14 -

any carbon source can be utilized, including lignocellulosics from agricultural by-products

while in practice, as for PLA and the other polyesters already discussed, further

improvements in fermentation yields by metabolic engineering of microorganisms, together

with technological advances in feedstock pre-treatment, such as new enzymatic processes,

are prerequisites for a shift to lower-value feedstocks.

PHAs have mechanical properties such as Young modulus and tensile strength resembling

those of thermoplastics like polyethylene and polypropylene (Matsusaki et al. 2000),

moreover, PHA is also biodegradable and bio-compatible.

Industrial production of poly-3-hydroxybutyrate (PHB) (Patnaik 2006), poly(3-

hydroxybutyrate-3-hydrox-yvalerate) (PHBV) (Jung et al. 2000) and poly(3-

hydroxybutyrate-3-hydroxy-hexanoate) (PHBHHx) (Chen et al. 2001) has been reported. By

careful control of the process they are encouraged to accumulate large quantities of PHB as

discrete intercellular granules. The transmission electron micrograph in Figure 1.9 shows a

thin section through a bacterial culture.

Figure 1.10. Transmission Electron Micrograph Showing Section Microbial Cells

Containing Discrete Granules of PHB (Magnification. 6300x)

The individual microorganisms can be clearly seen and each is full of white granules of

polymer. In fact up to 80% of the cell‘s dry weight can be in the form of PHB granules. The

polymer was first isolated and characterized in 1925 by Lemoigne at the Pasteur Institute in

Paris. Since then it has been extensively studied by biochemists that have come to the

general conclusion that bacteria store PHB as an energy reserve in much the same way that

mammals accumulate fat.

This polymer has unique physicochemical properties like thermoplasticity, biodegradability,

biocompatibility, and particularly its tensile properties, which are quite similar to that of

isotactic polypropylene (Vasile et al. 2003; Combeaud et al. 2004). Because of its properties,

PHB is useful in a wide variety of applications ranging from packaging to suture, bone

prostheses, and medicine capsules (Selke et al. 2006, Bucci et al. 2007). The use of PHB also

has limitations. One such limitation is its narrow processing window. The polymer is

thermally unstable at temperature above the melting point (about 180oC), and a drastic

reduction in molecular weight occurs during processing at temperatures between 180-200oC

Introduction

- 15 -

(Biresaw et al. 2002a). Another limitation of it is relatively low impact resistance. In order to

overcome these shortcomings, efforts have been made to co-polymerize PHB repeating units

with other monomers (Mohamed et al. 2007a, Biresaw et al. 2002b, Mohamed et al. 2007b,

Biresaw et al. 2008, Biresaw et al 2004, Sudesh et al 2000, Arrifin et al. 2008, Yang et al.

2004, Erceg et al. 2005), but the cost is considerably high, which limited its usage. One

possible approach to obtain a less expensive thermoplastic polymer of improved mechanical

properties is to blend PHB with another suitable polymer. Considerable work on blending

PHB with other polymeric matrix has been reported, indicating the total or partial miscibility

and the immiscibility of PHB with the second polymer. Earlier studies have shown that

blending polymers could lead to improve properties, decrease the cost and impart

functionalities in blends (De Carvaho et al. 2008).

For their versatility PHAs find application in many daily sectors such as packaging,

agriculture, furniture, transportation, textile, electric & electronic, houseware and biomedical

field (Philip et al. 2007).

1.4.2. Poly (lactic acid) (PLA)

In recent years, biodegradable polymers have been developed to become an attractive

alternative to petrochemical and chemosynthetic polymers. They offer upon conversion a

viable alternative to commodity plastics in a number of bulk applications where recycling is

impractical or uneconomical. Poly(lactic acid) (PLA) is a well-known biodegradable

thermoplastic polymer and appears to meet the safety environmental requirements (Iovino et

al. 2008, Tuominen et al. 2002).

Lactic acid can be produced by anaerobic fermentation of carbon based substrates such as

glucose, sucrose and starch, by using micro-organisms such as bacteria or certain fungi (Patel

et al. 2009, Johansson et al. 2012).

The physical and mechanical PLA properties make this polymer a good candidate as a

replacement of petrochemical thermoplastics in several application areas. While the PLA

high price long restricted its use to medical and specialty applications, recent breakthroughs

in lactide and polymerization technology opened up possibilities for the PLA production in

bulk volumes (Drumright et al. 2000).

Lactic acid may be produced by anaerobic fermentation of carbon substrates, such as

glucose, sucrose and starch, using micro-organisms such as bacteria or certain fungi.

The raw materials range suitable for lactic acid fermentation includes hexoses such as D-

glucose, together with a large number of compounds that can be easily split into hexoses

such as sugars, molasses, sugar beet juice, sulfite liquors and whey, as well as rice, wheat,

and potato starches.

In the future, it is expected that hydrolysis of lignocellulosics such as woody or herbaceous

biomass originating from wood, straw or corn stover, will become a viable pathway through

the enzymatic processes, together with the pressure on resources driving the increased

utilization of agricultural waste, thus avoiding the negative interference with food chain

resources.

The hardness, stiffness, impact strength and elasticity of PLA, are important for applications

such as beverage flasks, are similar to values for PET (Patel et al. 2009). It is a recyclable


- 16 -

and compostable material that provides significant energy savings (Drumright et al. 2000;

Bogaert et al. 2000).

PLA has a low vicat softening point so the obtained containers are not suitable for being

filled at elevated temperatures and exists the problem for the products warehousing and the

use in automobiles. On the other hand, PLA‘s low heat deflection temperature (HDT) and the

high heat seal strength lead to good performance in film sealing.

PLA has high odor and flavor barrier properties, high resistance to grease and oil, thus

finding application in the packaging of viscous oily liquids, dry and short shelf-life products.

It is not suitable for the packaging of carbonated beverages and other liquids due to its poor

O2, CO2 and water vapor barrier. This property is resulted interesting for some applications

such as the clothing sector where the high water transmission (high wick) for fabrics (Gruber

& O‘Brien, 2002) is a desirable characteristic. The hydrolytic stability conditions close to

some laundering, dyeing and finishing processes are borderline (Woodings, 2000).

PLA exhibits good chemical resistance to aliphatic molecules such as mineral oils and

terpenes.

Since PLA is a polar material it has a high critical surface energy and is thus easy to print,

metalize and dye. It is possible to print PLA using natural dyes and pigments that are heavy

metal free and thus eligible for the DIN norm compostable logo.

PLA is largely resistant to attack by microorganisms in soil or sewage at ambient

temperature, so the polymer must first be hydrolyzed at elevated temperatures (>58°C) to

reduce the molecular weight before biodegradation can commence. Thus, PLA will not

degrade in typical home composting, while it does break down in a typical industrial

composting facility.

Stereocomplex PLA is more resistant to biodegradation. Under typical use and storage

conditions PLA is quite stable. Additives which retard hydrolysis may be used for further

stabilization (Brandrup et al., 1999).

1.5. Different Media as Tool Boxes for Biodegradation of Plastic

Because of the low density of the majority of plastics, the volume fraction of plastics waste

in the municipal solid waste is by far greater than the weight fraction and plastics waste takes

up a lot of landfill space. Plastics rubbish, which makes up about 5±8 wt% of the municipal

solid waste, remains undegraded in the environment for such a long time that it causes

serious environmental problems. Therefore, a great deal of research has focused on

developing biodegradable plastics (Kim et al. 2000).

Biodegradation represents the key property for any form of material recycling or reuse and it

depends on the features of the disposal environment such as the moisture level, the surface

area and structure, the microbiological activity, and other nutrient materials.

Some researchers (Eubeler et al. 2010) reported methods such as standardized

biodegradation tests, analytical tests, enzymatic tests or tests of physical properties to

evaluate the biodegradability of synthetic and/or polymers for different types of

environmental compartments (e.g., soil, compost or river water).

The background used in performing biodegradation studies is constituted by a simple method

(Chiellini and Corti, 2003), the ASTM D5988–03, CEN/TR 15822/09 and EN 14046

Introduction

- 17 -

standards used to follow biodegradation experiment in soil medium, the ISO 14855, ISO

17088-12, EN 13432 standards for the analyses in mature compost and the EN ISO 14593

standard for experiments in river water.

1.5.1. PHAs Biodegradation in Soil

PHA degradation has been recorded in nature and attributed mainly to bacteria (Matavulj et

al. 2000, Lee 1996).

Fungi are another potent group of the degrading micro-biota and they may play an important

role in PHA breakdown, so only recently, fungal breakdown of PHA has attracted

considerable attention of many scientists (Rheddy et al. 2003, Sang et al. 2001, Sang et al.

2002, Garcia de Maria & Dominguez 2010).

One of the first indications of microbial, including PHAs fungal degradation in nature was

published by Lepidi et al. 1972 , who found that micro-fungi could be accumulated from

radioactive compounds originating from bacterially synthesized 14C labelled PHB.

Various traces, cavities, and grooves observed on the dented surface of PHBV films

demonstrated that the degradation was a concerted effect of a microbial consortium

colonizing the film surface, including fungi, bacteria, and actinomycetes.

The succession of microbial consortia in the soil around the PHBV films during the

degradation showed a distinctive increase in the fungal population, resulting in its

dominance. Comparison of the degradation ability of microbial strains isolated from soil

where PHBV films were degraded, revealed that fungi showed the highest contribution to

PHBV degradation, growing very rapidly along the film surface with their high degradation

ability and then expanding their hyphae in a three-dimensional manner (Sang et al. 2002).

Soil micro-organisms are able to degrade the BIOPOL component of blends with ethylene-

vinylacetate. In a few cases the enzymatic breakdown process of PHB by fungi has been

investigated in detail (Matavulj et al. 2000).

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs), which have constituents with a

typical chain length of C6-C14, are polyesters that are synthesized and accumulated in a

wide variety of Gram-negative bacteria, mainly Pseudomonas (Kim et al. 2007). mcl-PHAs

can be degraded by micro-organisms that produce extra-cellular mcl-PHA depolymerase.

mcl-PHA-degraders are relatively uncommon in natural environments and, to date, only a

limited number of mcl-PHA depolymerases have been investigated at the molecular level.

The ability to degrade PHAs depends on the secretion of specific extra-cellular PHA

depolymerases that hydrolyze the polymer to water-soluble products. These enzymes are also

divided into two groups, short-chain-length polyhydroxyalkanoates (scl-PHAS)

depolymerases and mcl-PHA depolymerases, which differ with respect to substrate

specificity for scl-PHAs or mcl-PHAs.

Although a few bacteria have been reported to degrade both scl-PHAs and mcl-PHAs by

producing two types of depolymerases (Klingbeil et al., 1996; Kim et al., 2003), the great

majority of PHA-degrading micro-organisms produce only one type of PHA depolymerase

that acts upon either scl-PHAs or mcl-PHAs.

Recently, Nam et al. 2002 described a list of taxa (60 different species belonging to 13

genera) of mcl-PHA degrading bacteria isolated from various soil samples and demonstrated


- 18 -

that Gram-negative bacteria belonging to the genera Stenotrophomonas and Pseudomonas

are the predominant mcl-PHA degraders in soil (Kim et al. 2007).

Degradation of polyhydroxyalkanoate (PHA) by three Pseudomonas sp., isolated from fuel-

oil contaminated soil and identified as Ps. fluorescens, Ps. aeruginosa and Ps. putida on the

basis of 16S rRNA sequence analysis, was investigated by Colak and Guner 2004. This

property was determined as molecular weight loss measured by gel permeation

chromatography and morphological changes in poly-3-hydroxybutyrate films observed by

scanning electron microscopy. Both, decrease in molecular weight (7–17%) and weight loss

(up to 29%) of solution-cast films were evidence of the isolates secreting an active

depolymerase responsible for degradation of PHA. Stimulation of activity by Na+, K+, Ca2+

and Mg2+ at 1 mM concentration indicated a requirement for metal ions as a cofactor for

activity. Inhibition of the extra-cellular depolymerases of the three strains in the presence of

p-methylphenyl sulfonylfluoride, cyanide, azide, deoxycholate or EDTA confirms the

presence of an enzyme in the isolates similar to poly(3-hydroxybutyrate) degrading

hydrolases (Colak and Guner 2004, Jendrossek 2001, Champrateep 2010).

A selected polyhydroxyalkanoate (PHA) film was evaluated (Srideshi et al 2006, Yew et al.

2006) for its degradation trends, in a tropical mangrove environment or in tropical climate

environment (Akmal et al. 2003). The biodegradability of PHB and its co-polymers PHBV

[P (3HB-co-5 mol% 3HV)] or PHBHHx [P(3HB-co-5 mol% 3HHx)], was investigated along

with PHB films containing 38 wt% titanium dioxide (TiO2) [PHB-38 wt% TiO2]. The

degradation of these formulations was monitored for 8 weeks at three different zones in an

intermediate mangrove compartment along Sungai Pinang, adjacent to a famous fishing

village on south of Penang Island. The degradation rate was observed both on the surface and

in the sediment and it was expressed in percentage of weight loss. The microbial

enumeration done using sediment from the different zones indicated similar colony-forming

unit (CFU) counts even though differences were noticed in the degradation profile of the

various films in the respective zones.

The results obtained revealed that co-polymers disintegrated at similar or higher rate than the

homopolymer, PHB. However, the incorporation of TiO2 into PHB films caused the

degradation rate of PHB -38 wt% TiO2 composite film to be far slower than all the other

PHA films. The overall rate of degradation of all PHA films placed on the sediment surface

was slower than those buried in the sediment. Microscopic analyses showed that the surface

morphology of P (3HB-co-5 mol% 3HHx) was more porous compared to PHB and P (3HB-

co-5 mol% 3HV) films, which may be an important factor for its rapid degradation.

The biodegradation of films made of poly-β-hydroxybutyrate (PHB) with a molecular mass

of 1500 kDa was studied by Bonartseva et al. 2002 using a model soil community in the

presence and absence of nitrate and at different concentrations of oxygen in the gas phase.

The PHB biodegradation was investigated with respect to changes in its molecular mass,

crystallinity, and some mechanical properties, surface composition and morphology (Sadi et

al. 2010, Lucas et al. 2008, Correa et al. 2008). The multi-block copolymers has been

evaluated by Zhao et al. 2006 using a soil suspension system.

Mergaert et al. 1992 isolated from soil more than 300 microbial strains capable of degrading

PHB in vitro, of which denitrifying bacteria are of particular practical interest due to their

Introduction

- 19 -

potential ability to be used in the immobilized state on PHB films for the purification of

water from nitrates.

The PHB film biodegradability was studied under aerobic, micro-aerobic and anaerobic

conditions (Maergaert et al. 2011) in the presence and absence of nitrate by microbial

populations of soil, sludges from anaerobic and nitrifying/denitrifying reactors, and sediment

of a sludge deposit site, as well as to obtain active denitrifying enrichment culture degrading

PHB.

Changes in molecular mass, crystallinity, and mechanical properties of PHB have been

studied.

The biodegradation of PHB films in soil suspension has been studied during 2 months under

different aeration conditions and nitrate was added as an additional nitrogen source (and as

an electron acceptor). PHB degradation was highest under aerobic condition, and practically

did not proceed under anaerobic condition. The addition of nitrate enhanced the degradation

under all of the aeration conditions studied and stimulated the development of denitrifying

microbial populations.

The presence of nitrate promoted the degradation and enhanced the decline in the Mw of

PHB films. The most degraded PHB exhibited the highest values of the crystallinity index.

As it has been shown by Spyros et al. 1997, PHAs contain amorphous and crystalline

regions, of which the former are much more susceptible to microbial attack. If so, the PHB

microbial degradation must be associated with a decrease in its molecular weight and an

increase in its crystallinity.

The total number of micro-organisms on the surface of the PHB films degraded under micro-

aerobic and aerobic conditions in soil was 4 x 104 to 5 x 106 (fungi) and 1 x 105 to 2 x 107

(bacteria). The PHB film degraded under anaerobic conditions contained no micro-

organisms. The bacteria detected on the degraded PHB films were dominated by the genera

Pseudomonas (Shimao 2001, Bhatt et al. 2008), Bacillus, Azospirillum, Mycobacterium,

Streptomyces (Hermida et al. 2009, Kulkarni et al. 2011, Rai et al. 2011), Actinomadura sp

(Shah et al. 2010) while the fungi were dominated by the genus Penicillium.

The microbial degradation of PHB tensile test pieces or its copolymer made of 3-

hydroxybutyric acid (90%) and 3-hydroxyvaleric acid (10%) was studied in soils by

Mergaert et al. 1993. The samples were incubated at a constant temperature of 15, 28, or

40°C for up to 200 days and the degradation was measured through loss of weight (surface

erosion), molecular weight, and mechanical strength. The erosion rate was enhanced by

incubation at higher temperatures, and in most cases the copolymer lost weight at a higher

rate than the homopolymer. The molecular weight decrease is due to simple hydrolysis and

not to the action of biodegrading micro-organisms.

The degradation resulted in loss of mechanical properties (Mergaert et al. 1993).

The number of denitrifying bacteria was about 102 cells per flask in almost all experimental

variants (Bonartseva et al. 2002).

PHB biodegradation in the enriched culture obtained from soil on the medium used to

cultivate denitrifying bacteria has been also studied. The dominant bacterial species,

Pseudomonas fluorescens and Pseudomonas stutzeri, have been identified in this enrichment


- 20 -

culture. Under denitrifying conditions, PHB films were completely degraded for seven days.

Both the film weight and Mw of PHB decreased with time.

The data were in contrast with those of Doi et al. 1990 who found that Mw of PHB remained

unchanged upon enzymatic biodegradation in an aquatic solution of PHB- depolymerase

from Alcaligenes faecalis. The average viscosity of the higher- and lower-molecular weight

polymers decreased gradually from 1540 to 580 kDa and from 890 to 612 kDa, respectively.

The "exo"-type cleavage of the polymer chain, such as the successive removal of the

terminal groups, is known to occur at a higher rate than the "endo"-type cleavage, such as the

random breakage of the polymer chain at the enzyme-binding sites. Thus, the former type of

polymer degradation is primarily responsible for changes in its average molecular weight.

Biodegradation of the lower-molecular polymer, which contains a higher number of terminal

groups, is more active, probably, because the "exo"-type degradation is more active in lower

than in higher molecular polymer (Bonartseva et al. 2002, Bonartseva et al. 2003, Doi et al.

1997).

The PHB samples have been tested for fungicidity and resistance to fungi by estimating the

growth rate of test fungi from the genera Aspergillus, Aureobasidium, Chaetomium,

Paecilomyces, Penicillum, Trichoderma under optimal growth conditions. PHB film did not

exhibit neither fungicide properties, nor the resistance to fungal damage, and served as a

good substrate for fungal growth (Mokeeva et al. 2002).

The degradability of poly(propylene carbonate)/poly(β-hydroxybutyrate-co-β-

hydroxyvalerate) (PPC/PHBV) blends has been investigated in soil suspension cultivation

and in vitro degradation testing by Tao et al. 2009. The changes of structure and molecular

weight for blends have been also studied by 1H nuclear magnetic resonance spectroscopy

(1H-NMR), scanning electron microscopy (SEM) and gel permeation chromatography

(GPC) before and after degradation. Although the PPC/PHBV blends were immiscible, the

addition of PHBV could improve the thermal stability of PPC. PHBV has been degraded

mainly by the action of microbial enzymes in the soil suspension, which biodegraded it more

rapidly than PPC in a natural environment.

PPC has been degraded mainly by chemical hydrolysis and random hydrolytic scission of

chains in the PBS solution in vitro, and degradation of PPC was more rapid than that of

PHBV in a simulated physiological environment.

Poly (lactic acid) degradation is a complex phenomenon characterized by morphological

changes correlated with the soil conditions.

The biodegradation propensity for flax fiber reinforced PLA based composites in presence of

amphiphilic additives was investigated by using the soil burial test (Kumar et al. 2010). PLA

removal from the composites surface depends on biodegradation as evidenced in SEM

study.. About 4.2% weight loss was observed in neat PLA of burial in soil for 20 days. After

90 days, composite samples partially disintegrated.

Poly (L-lactide) (PLLA) films were buried in outdoor environment for two years for testing

the biodegradability as Gallet et al. 2001 reported. After 20 months lactic acid and lactoyl

lactic acid appeared as the products of hydrolysis. After 24 months the amount of lactoyl

lactic acid decreased due to the biotic hydrolysis of the film. So PLLA was degraded by a

combination of hydrolysis and biotic activity. In first stage, after an induction period of 1

Introduction

- 21 -

year, PLLA film undergoes hydrolysis, while in second stage microorganisms assimilate the

small degradation products also hydrolysis happened. The thermal properties (Tg and Tm)

were significantly affected during the period (Karjomaa et al. 1998, Hakkarainen et al. 1996,

Hakkarainen et al. 2000a and 2000b).

Soil biodegradation were performed experiments by using 2mm thick racemic PLA plates

during 8 weeks (Torres et al. 1996).

Although no significant changes were observed in PLA oligomers concentration, lactic acid

was removed from the film by microorganisms‘ assimilation while hydrolysis formed large

amount of them. Thus both hydrolysis and microbial assimilation affect PLLA degradation at

same time.

The long-term degradation/disintegration behavior of PLA films and fibres, with eleven-

month trial, was investigated, in the experimental field of natural Mediterranean soil, by

Rudnik et al. 2011. Different thicknesses for PLA films, are responsible of mechanical

degradation or partially disintegration.

The mechanical, thermal and biodegradable properties of poly [(D, L-Lctide)] (PDLLA),

together with other polymers (PEG- poly (ethylene glycol)) were assessed by Wang et al.

2008.

The degradation test showed that PEG addition could accelerate the blends biodegradation in

soil under room temperature significantly, and proximately 20 % mass loss was got by the

end of 30 days by means of storage.

Shogren et al. 2003 found that burning PLA in soil for 1 year, from outside to inside along a

narrow zone, weight losses were due to starch only with the degradation proceeding.

1.5.2. PHAs Biodegradation in Mature Compost

PHA production and degradation in waste environment as well as its role in biological

phosphorus removal were reviewed by Lee et al. 1999. In biological phosphorus removal

process, bacteria accumulating polyphosphate (polyP) uptake carbon substrates and

accumulate these as PHA by utilizing energy from breaking down polyP under anaerobic

condition. In the following aerobic condition, accumulated PHA is utilized for energy

generation and for the regeneration of polyP. PHA production from waste has been

investigated in order to utilize abundant organic compounds in waste water. Since PHA

content and PHA productivity that can be obtained are rather low, PHA production from

waste product should be considered as a coupled process for reducing the amount of organic

waste. PHAs can be rapidly degraded to completion in municipal anaerobic sludge by

various micro-organisms.

Aiba et al. 2009 have isolated a thermophilic Streptomyces sp., capable of degrading various

aliphatic polyesters, from a landfill site. The isolate, Streptomyces sp. BCC23167,

demonstrated rapid aerobic degradation of several polyesters, including

polyhydroxyalkanoate copolymers, poly(ε-caprolactone) and poly(butylene succinate) at

50◦C and neutral pH. The degrading activity was repressed by glucose and cellobiose, but

tolerant to repression by other carbon substrates.

Degradation of a commercial poly [(R)-3-hydroxybutyrate-co(R)-3-hydroxyhexanoate]

(PHBHHx) by Streptomyces sp. BCC23167 progressed from surface to bulk as suggested by

the slight decrease in polymer molecular weight. Differential scanning calorimetry analysis

of PHBHHx film degradation by Streptomyces sp. BCC23167 showed that relative


- 22 -

crystallinity of the film increased slightly in the early stage of degradation, followed by a

marked decrease later on. The surface morphology of degraded films was analyzed by

scanning electron microscopy, which showed altered surface structure consistent with the

changes in crystallinity.

The isolate is thus of potential interest for application in composting technology for

bioplastic degradation (Rheddy et al. 2003, Weng et al. 2010, Weng et al 2011).

A Gram-positive poly (3-hydroxybutyrate) (PHB)-degrading bacterial strain was isolated

from compost by Takaku et al. 2006. This organism, identified as Bacillus megaterium N-18-

25-9, produced a clearing zone on opaque PHB agar, indicating the presence of extracellular

PHB depolymerase. A PHB depolymerase gene, PhaZBm, of B. megaterium N-18-25-9 was

cloned and sequenced, and the recombinant gene product was purified from Escherichia coli.

The N-terminal half region of PhaZBm shared significant homologies with a catalytic

domain of other PHB depolymerases.

Also the PHA-degrading isolate, strain P37C, was enriched from residential compost for its

ability to hydrolyze the medium chain length PHA, poly(β-hydroxyoctanoate) (PHO). It was

subsequently found to grow on a wide range of PHAs, including both short chain length and

medium chain length PHAs. The isolate was identified as belonging to the genus

Comamonas. Strain P37C formed clear zones on PHB, PHO and poly(β-

hydroxyphenylvalerate) (PHPV) overlay plates (Quinteros et al. 1999).

PLA blends are degradable under both aerobic and anaerobic conditions, indicating that

composting, landfilling and anaerobic digestion represent all possible options for handling

the waste. However, pretreatment technologies, such as thermal, enzymatic, ultrasonic

treatment and the application of adapted microorganisms, must be combined with

composting in order to enhance the reduction of the waste volume (Cho et al 2011, Zeid et al.

(2001).

Biodegradation rates of PLA/starch blends and PLA/wood- flour composites were lower than

pure cellulose but higher than pure PLA. This value increased from 60% to 80% when the

starch content was increased from 10% to 40% after 80 days (Kunioka et al. 2006). It means

that an higher starch content makes faster PLA biodegradation in composting facilities.

Many authors have prepared PLA based nano-composites and studied several properties

including biodegradability in composting.

Bacillus licheniformis could be considered as one of the responsible bacterium for PLA

biodegradation in compost. The influence on the chemical structure as the interaction of the

bacterium toward clay, are clear effects exerted on the polymer bacterial degradation.

In the case of 4% by weight of filler content, the silicate layers were homogeneously

dispersed in the PLA matrix and these hydroxy groups start heterogeneous hydrolysis of the

PLA matrix after absorbing water from compost. The weight losses and PLA hydrolysis

degree for the pure sample and the composite containing 4% by weight of filler remained

almost same up to one month that became a critical time for PLA degradation rate (Pandey et

al. 2005).

Poly (l-lactide) (PLLA) could rapidly biodegrade under mixed culture of compost

microorganisms‘ conditions (Hakkarainen et al. 2001, Nampoothiri et al. 2010). The films

was fragmented to fine powder and analyzed after 5 weeks in biotic environment. The

Introduction

- 23 -

performed analysis in the biotic environment showed that microorganisms assimilated lactic

acid and lactoyl lactic acid from the films rapidly. At the same time, a new degradation

product that is an ethyl ester of lactoyl lactic acid was formed and a rapid molecular weight

and polydispersity increase was observed.

The SEM micrographs results showed a large number of bacteria and mycelium of fungi

growing on the surface of the biotical aged films. On the other hand, in the abiotic

environment only a slight molecular weight decrease was observed and the polydispersity

decreased towards 2.0.

Moreover, preferred degradation happened near the chain ends in the biotic environment

whereas a random hydrolysis of the ester bonds in abiotic environment occurs.

The compostability of three commercially available biodegradable packages made of PLA,

in particular water bottles, trays and deli containers, to composting and to ambient exposure

were studied by Kale et al. 2006 that investigated the properties breakdown of these

packages exposed to compost conditions by several experimental procedure involving GPC,

DSC, TGA and visual inspection. The compost pile was prepared with cow manure, wood

shaving and waste feed, at a temperature above 60°C. After 3 weeks the pile was put on

asphalt pad. The initial pile temperature, relative humidity and pH was 65 ± 5 °C, 63 ± 5%

and 8.5 ± 0.5, respectively.

The packages were subjected to composting for 30 days and the packages exposed to

ambient conditions were also studied. This is because it is well known that PLA absorb

water, resulting in the hydrolysis and cleavage of the ester linkages, auto-catalyzed by the

carboxylic acid end groups.

Kale et al. (2006) found that the degradation rate changes with the initial crystallinity and L-

lactide content of the packages, with lower degradation for the polymer with the highest

content of L-lactide (96 versus 94%) due to the higher crystallinity, which makes more

difficult the degradation of the whole structure. During the hydrolysis process the sample

decreased in size and became tough with a trend that depends to the shape.

The dimension of the containers before and after composting was calculated by measuring

the variation on width, length, height and thickness. During the first period of the ambient

exposure (within the first 15 days) the molecular weight of the sample Mw showed a small

increase probably due to UV or gamma radiation that produce chain cleavage and subsequent

recombination, which can result in cross-linking and hence an increase in the Mw. The

increase in molecular weight produced an increase in the Tg and leading to slower

degradation since glassy polymers degrade more slowly than rubbery ones. The same trend

was observed for the sample exposed to the compost pile; the Mw increase could be

attributed to cross-linking or recombination reactions. Subsequently, during exposure, the

degradation produced a molecular weight decrease that followed a first order kinetic

associated to a first order hydrolysis process affected by the initial crystallinity, thickness

and shape of the sample (Tsuji et al. 1998).

1.5.3. PHAs Biodegradation in Aqueous Medium

Fungi were probably involved in the degradation of BIOPOL shampoo bottles under marine

conditions. More recently, fungal breakdown of thermoplasts, including PHA has been

simulated in the deep sea conditions.


- 24 -

BIOPOL (Zeneca BioProducts) (Matavulj et al. 2000), a copolymer of PHB and PHV, has

been commercially produced and is used as packaging material. In vitro bacterial degradation

of BIOPOL and several other PHAs has been proven (Fedulova et al. 1980, Tanio et al.

1982, Jendrossek et al. 1992, Jendrossek et al. 1996, Schirmer et al 1993).

In addition to the type of micro-biota involved, the rate of biodegradation depends also on

many other factors, notably those related to the ease of microbial colonization.

Environmental factors, such as humidity, temperature, electrochemical reaction, biological

oxygen demand and supply of other nutrients (nitrogen, phosphorus), are also important.

The BIOPOL degradation (Matavulj et al. 2000) as well as its blends degradation with other

plastic materials such as poly(caprolactone), polystyrene, poly-L-lactide and polystyrene-co-

acrylonitrile can be performed by using PHA depolymerase from the culture supernatant of

the fungus Penicillium funiculosum.

The tested blends showed higher weight losses with increasing content of the BIOPOL

component.

Doi et al. 1999 have evaluated the biodegradabilities of (P(3HB-co-14%3HV)) monofilament

fibers at 25°C for 28 days by monitoring the time-dependent changes in the biochemical

oxygen demand (BOD) and weight-loss (erosion) of fibers under aerobic conditions in a

temperature-controlled reactor containing natural waters from various aquatic environments

such as sea, lake and river (Rheddy et al. 2003, Luzier et al. 1992, Khanna & Srivastava

2005).

Two types of fibers with different diameters (213 and 493μm) were used in this test and their

biodegradabilities decreased in the following order: river > lake > sea. By analyses of

scanning electron microscopy and X-ray diffraction of eroded fibers, it has been concluded

that the biodegradation proceeded from amorphous regions on the surface of fibers by the

function of micro-organisms in fresh water or seawater. In addition, 13 strains of PHB-

degrading bacteria were isolated from different sources of seawater and identified. Majority

of isolates grew well on PHB or P(3HB-co-14%3HV) as sole carbon source and excreted

extra-cellular polyhydroxybutyrate (PHB) depolymerases (Ohura et al. 1999).

During the summers of 1999-2001 the dynamics of polyhydroxyalkanoate degradation in a

small recreational eutrophic reservoir or in anaerobic sediment (Castelli et al. 1995) was

studied (Volova et al. 2006, Volova et al. 2007, Volova et al. 2010).

It has been shown that biodegradation of polyhydroxyalkanoates in the environment is

determined by the structure and physicochemical properties of the polymer and by local

weather conditions, which influence the state of the aquatic ecosystem. Species (clones) of

bacteria able to utilize polyhydroxyalkanoates in the reservoir were identified using

molecular phylogenetic analysis of 16S rRNA genes.

The attempts to examine the abundance and diversity of mcl-PHA-degrading micro-

organisms in marine environments showed that mcl-PHA degraders in seawater samples

were less than 1.4% of all the heterotrophic bacteria and that Pseudomonas and

Stenotrophomonas strains accounted for approximately 65% and 23%, respectively, of the

total mcl-PHA degraders isolated (Kim et al. 2007, Doi et al. 1992). These results suggest

that mcl-PHA biodegradation in soil and marine environments may be predominantly

performed by Pseudomonas and Stenotrophomonas species. Among Gram-positive bacteria,

Introduction

- 25 -

the presence of mcl-PHA depolymerase activity has been found only in Streptomyces sp.

(Klingbeil et al., 1996; Kim et al., 2003) and Rhodococcus equi (Lim, 2006). Particularly,

members of the genus Rhodococcus have attracted great interest due to their capabilities to

degrade various hydrophobic substances such as petroleum hydrocarbon, benzene,

polychlorinated biphenyls, and scl-PHAs (Jang et al., 2005; Taki et al., 2007; Valappil et al.,

2007). The ability of Rhodococcus to degrade mcl-PHAs suggests that Rhodococcus may

play a considerable role in decomposing various materials in natural environments (Kim et

al. 2007).

Biodegradabilities of 21 samples of biosynthetic and chemosynthetic polyesters were

measured at 25°C (Doi et al. 1996, Kasuya et al. 1998, Ohura et al. 1999), under aerobic

conditions in a temperature-controlled reactor containing 200 ml of natural water from the

river Arakawa (Saitama, Japan) as an inoculum, by monitoring the time-dependent changes

in the biochemical oxygen demand (BOD), weight loss (erosion) of polyester film and

dissolved organic carbon concentration (DOC) of the test solution. The microbial

copolyesters, such as PHBV, poly(3- hydroxybutyrate-co-4-hydroxybutyrate) and poly(3-

hydroxybutyrate-co-3- hydroxypropionate), were degraded in the river water at a rapid rate,

and the weight-loss and BOD-biodegradabilities of the majority of biosynthetic polyesters

were 100 and 80% ± 5% for 28 days, respectively (Doi et al. 1996).

The same polyesters have been studied (Abou-Zeid et al. 2001, Hablot et al. 2008) in two

anaerobic sludges and individual polyester degrading anaerobic strains were isolated,

characterized and used for degradation experiments under controlled laboratory conditions.

Two methods for accurate poly (3-hydroxybutyrate) (PHB) depolymerase activity

determination and quantitative and qualitative hydrolysis product determination are

described by Gebauer and Jendrossek 2006. The first method is based on online

determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid

(3HB) and/or 3HB oligomer produced during the hydrolysis reaction and requires a pH-stat

apparatus equipped with a software controlled microliter pump for a rapid and accurate

titration. The method is universally suitable for hydrolysis of any type of

polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the

determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at

least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB

granules isolated from Ralstonia eutropha H16 were determined for the first time. The

second method was developed for hydrolysis product identification and is based on the

derivatization of 3HB oligomers into bromophenacyl derivates and separation by high

performance liquid chromatography. The method allows the separation and quantification of

3HB and 3HB oligomers up to the octamer.

The two methods were applied to investigate the hydrolysis of different types of PHB by

selected PHB depolymerases (Gebauer and Jendrossek 2006).

An effective technology for production of PHB and PHBV of different molecular weight

(from 200 to 1500 kDa) by diazotrophic bacteria of Azotobacter and Rhizobium genus has

been developed (Bonartsev et al. 2007). In order to clarify mechanism of PHB

biodegradation degradation of PHB at different conditions in vitro and in vivo have been

studied.


- 26 -

The mechanisms of PHB biodegradation is correlated with a PHB hydrolysis in vitro

(Shangguanna et al. 2006). Degradation of films from PHB of different Mw (300, 450, 1000

kDa) in vitro has been demonstrated under three different conditions that showed

simultaneously the increase of crystallinity and the decrease of Mw. As consequence,

mechanical properties became worse: PHB films became more rigid and fragile; the surface

of PHB films became distended, micro-cracks appeared. At second relatively short-term

phase rapid fragmentation and following complete degradation of small fragments of PHB

films were observed.

PHB degradation by activated sludge from reactor treating pig manure wastewater and from

nitrifying/denitrifying reactor of the plant treating municipal wastewater (Moscow) in the

presence and absence of nitrate at 20°C has been studied (Kallistova et al. 2002, Hong et al.

2008, Chen & Li 2008, Wang et al. 2004). The activated sludge from both reactors had

similar properties for PHB degradation and average rates of denitrification. In both presence

and absence of nitrate, the intensity and properties of PHB degradation were also similar.

The rate of PHB degradation in the presence of nitrate was only 1.11 times higher, than in

absence of nitrate. The activated sludge from anaerobic reactor showed a bit higher average

rates of denitrification and acetate accumulation.

The contribution of bacteria, isolated from sewage sludge, towards the microbial degradation

of PHBV was studied by Shah et al 2007.

A microbial consortium (De Lucas et al. 2005) was inoculated on mineral salt agar plates

containing different concentrations of PHBV as a substrate. The PHBV degraders showed

optimum depolymerase production at pH 7.0, 37°C, and 0.4% substrate concentration during

2–5 weeks of incubation. Additional carbon sources like glucose, fructose, sucrose, lactose

and nitrogen sources including yeast extract were used as co-metabolites. The highest degree

of degradation of PHBV was observed in the presence of glucose. When the PHBV

depolymerase activity of isolates present in the consortium was determined separately, it was

found that the enzyme activity was higher in case of Bacillus sp. AF3. The purification steps

included ammonium sulphate fractionation and gel permeation chromatography on Sephadex

G-75. The enzyme has been purified to homogeneity as judged by sodium dodecyl sulphate-

polyacrylamide gel electrophoresis (SDS-PAGE) and molecular weight was found to be

approximately 37 kDa (Shah et al. 2007).

The hydrolytic degradation of poly(3-hydroxyalkanoates), PHA blends with low and high

molecular weight additives have been examined by Renarda et al. 2004. The PHA films were

characterised by Differential Scanning Calorimetry, DSC, and Atomic Force Microscopy,

AFM, which revealed that hydrolyzable PLA and hydrophilic PEG, selected as additives

which might accelerate hydrolytic degradation, were immiscible with PHAs over the range

of compositions studied. PHA blends were incubated in tetraborate buffer (pH 10) at 37°C

for periods ranging from 7 to 160 days.

Film degradation was studied by weight loss and Size Exclusion Chromatography (SEC).

The results showed that the presence of a second component, whatever its chemical nature, is

sufficient to perturb the crystallization behaviour of highly crystalline, PHBV, and increase

hydrolytic degradation. In contrast, the degradation of poly(3-hydroxyoctanoate), PHO, was

unaffected by blending with PLA or PEG. PHO degradation is a very slow process, requiring

Introduction

- 27 -

several months of incubation. However, the introduction of polar carboxylic groups in side

chains led to an increase in the degradation rate.

PLLA mineralization (Reeve et al. 1994), in the aerobic aquatic headspace test, was very

slow at room temperature showing that its polymer structure has to be hydrolysed before

microorganisms can utilize it as a nutrient source so, as occurs for other

polyhydroxyalkanoic acids (Jendrossek et al. 1996), PLLA crystalline structure reduces

chemical hydrolysis and therefore its susceptibility to biodegradation.

When PLLA is exposed to high temperatures, fundamental microstructural changes and

molecular rearrangements occur and hydrolysis starts (Itavara et al. 2002). The flexibility of

the polymer chain increases at temperatures higher than the glass transition temperature. In

addition, water absorption into the polymer matrix increases at high temperatures. This may

not only accelerate chemical hydrolysis, but also facilitate the attachment of microbes and

enzymes on the polymer surface by increasing polymer hydrophilicity.

The molecular size of the PLLA oligomers that microorganisms are able to utilise is critical,

as reported in our earlier study (Karjomaa et al. 1998).

1.5.4. Microbial Biodegradation Mechanism (intra and extra-cellular)

Deep studies haven been conducted recently by Hidayah Ariffin et al 2008 and 2009 about

the thermal degradation mechanisms of PHB. They confirmed that random chain scission by

β-elimination is the main mechanism of PHB thermal degradation. The authors previously

have been suggested the possibility of additional mechanism where an auto-accelerated

random degradation has been favored by the carboxylic groups self-proliferated in bulk. The

unzipping reaction occurring at the end of the molecules was kinetically favored by the

scission of polymer chains that generates a large number of crotonic acid moieties. The

author suggested that this is an ideal self-proliferation reaction of carboxyl compounds and is

the key factor in the auto-accelerated random degradation. An upgrading of this study

conducted by Ariffin et al., to hypothesize a new mechanism for PHB thermal degradation

(Scheme 1.1) where anhydrides production has been proposed as an additional pyrolysis

mechanism of the biopolymer.

O CH2

C

O

CHO

H2CH

CH2

C

O

CHO

CH3

O CH2

C

O HC

CH2+

CH2

C

O

CHO

H3C

Scheme 1.1. Thermal PHB-chain Breakdown with Formation of Crotonic and Carboxilic

End Groups.


- 28 -

Moreover, they observed a wide range of activation energy value (Ea) for the PHB thermal

degradation and they hypothesized that probably this wide range can be correlated with a

deviation from the random chain scission statistics suggesting the presence of other kinds of

mechanism out of the random scission. To confirm other mechanisms out of the random

scission, minor pyrolyzates from PHB were characterized with 1H/13C-NMR, Fourier

transform infrared spectroscopy, and Fast Atom Bombardment Mass Spectrometry. As a

result, crotonic anhydride and its oligomers were detected as minor products from

condensation reactions between carboxyl groups. The anhydrides production must be one

reaction out of the conforming process to the random degradation statistics and contribute to

the complexity of PHB pyrolysis. Then, an expected thermal degradation pathway of PHB

was proposed (Scheme 1.1) (Arrifin et al. 2009).

Another mechanism that gives a decreasing in (PHA)s molecular weight is the hydrolytic

degradation (Scheme 1.2) promoted by the introduction of polar carboxylic groups in side

chains.

Scheme 1.2. PHB-Chain Breakdown by Moisture as Auto-catalyzed by Carboxilic End

Groups.

Renarda et al. 2004 characterized PHAs films by differential scanning calorimetry and

atomic force microscopy finding that hydrophilic components such as PLA and PEG might

accelerate the hydrolytic degradation correlated with a perturbation in the crystallization

behaviour of highly crystalline PHBV.

The hydrolytic degradation of PHB proceeds homogeneously and randomly via a bulk

erosion mechanism that proceed faster in the melt than in the solid state. The molecular

weight of PHB decreased exponentially without formation of low molecular-weight specific

peaks originating from crystalline residues (Saeki et al. 2005).

O CH2

C

O

CH

CH3

O CH2

C

O

CH

H3C

O

O CH2

C

O

CHOH

CH3 HO

CH2

CH

O

CH3

H3O

+ H

CO

+

H2O

Introduction

- 29 -

The activation energy for the hydrolytic degradation of PHB in the melt (180–250°C) was

30.0 kcal /molK, which is higher than 12.2 kcal/molK for PLLA in the melt in the

temperature range (180–250°C). So using this method is possible to obtain (R)-3HB and to

prepare PHB having different molecular weights without containing the specific low-

molecular weight chains, because of the removal of the effect caused by crystalline residues.

The molecular weight distribution of PHB shifted as a whole to a lower molecular weight

during the hydrolytic degradation without formation of any low molecular- weight specific

peaks arising from the chains in the crystalline residues. No formation of any low-molecular-

weight peaks is an important feature of the hydrolytic degradation of PHB above Tm, which

must contribute to its efficient hydrolytic degradation, resulting in rapid saturation of yields

of 3HBs.

The Mw/Mn value remained in the range of 1.5–1.8 during the hydrolytic degradation as

long as Mn was higher than 1x 104 g/ molK, while it increased to about 2.7 when Mn

became lower than 1x 104 g/ molK. The Mn decreased exponentially with the hydrolytic

degradation time and the decrease rates of Mn increased monotonically with the hydrolytic

degradation temperature.

1.5.5. PHAs Biodegradation Provided by Enzymes

PHA depolymerase obtained from some fungi or bacteria such as Penicillium funiculosum,

Alcaligenes faecalis, Pseudomonas leimoignei are a class of enzymes suitable for PHAs

degradation and its based blends (Hisano et al. 2006, Nojiri et al. 1997, Iwata et al. 2002).

Higher weight losses with increasing BIOPOL component were obtained. Similar results

were reported (Matavulj et al. 2000) by other researchers who assessed that only the

BIOPOL component of blends with ethylene-vinylacetate was degraded by soil

microorganisms (Verhoogt et al. 1995).

Enzymatic degradation of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) (PHBHA) bio-

polyesters consisting of HB and 15 mol % medium-chain-length HA was studied using the

enzyme PHA depolymerase produced by Ralstonia pickettii T1. It was found that PHBHA

films did not loose their weight after 25 h of depolymerase treatment. In contrast, three

commercially available PHAs including PHB, poly (3-hydroxybutyratee19 mol % 3-

hydroxyvalerate) (PHBV) and poly (3-hydroxybutyrate - 19 mol % 3-hydroxyhexanoate)

(PHBHHx) lost over 50% of their original weights. Slow degradation of PHBHA was also

confirmed by the absence of HA monomers, dimers or trimers as degradation products in

their depolymerase solution compared with abundance of degradation products released by

the other three PHAs under the same condition (Li et al. 2007, Hiraishi et al. 2006).

The photocatalytic activity and biodegradation of PHB films containing titanium dioxide

(TiO2) were evaluated. PHB-TiO2 based films (Yew et al. 2006) and after an hour under

solar illumination in garden soil, 96% of methylene blue solution was decolorized.

The antibacterial activity against Escherichia coli (E. coli) exhibited enhanced photocatalytic

sterilization activity over time. PHB-TiO2 based films showed slower degradation rate in

dark conditions than those receiving direct solar illumination. Interestingly, the latter

composite films showed faster degradation rates compared to pure PHB films indicating that

the degradation is mainly due to photocatalytic activity. PHB-TiO2 based films buried in soil


- 30 -

generally showed slower degradation rates than pure PHB films. These values were

dependent on the soil microbial activity.

Poly (lactic acid) depolymerases can be categorized into protease-type and lipase- type

(Kawai et al. 2011). During PLA hydrolysis process, the protease enzymes are selective for

PLLA whereas the lipase enzymes, including Cutinase-Like Enzyme (CLE) from

Cryptococcus sp. strain S-2 preferentially hydrolyze PDLA. Both enzymes degraded not only

PLA emulsion but also PLA film, in which amorphous region is preferentially attacked,

respect the crystalline crystalline one.

The different enantio-selectivity of these active enzymes toward PLLA and PDLA could be a

good indicator for categorizing PLA diastereoisomers.

Microorganisms such as Fusarium moniliforme and Penicillium Roquefort that carry out the

degradation of PLA oligomers (molecular weight ~1000) (Torres et al. 1996) or

Amycolatopsis sp and Bacillus brevis are able to degrade PLA polymer (Pranamuda et al.

1997, Pranamuda et al. 1999, Tomita et al. 19999. In addition, enzymatic degradation of low

molecular weight PLA (molecular weight ~2000) has been shown using esterase-type

enzymes such as Rhizopus delemer lipase (Fukuzaki et al. 1989).

Kunioka et al. 2006 have patented some methods with the aim to produce mechanical

crushing of PLA pellets and powder at low temperature, by using selected dry ice. The

biodegradation rate of these PLA based powders were evaluated with two methods:

enzymatic degradation and Microbial Oxidative Degradation Analyzer (MODA) technique,

both based on an international standard.

In the first case, PLA biodegradation degree was 91% for 35 days in a controlled compost

according to a protocol based on ISO 14855-1 (JIS K6953) at 58ºC whereas in the second

case, the PLA biodegradation degree was almost 80% for 50 days.

Proteinase K., a fungal serine protease of Tritirachium album (Williams 1982, Tomita et al.

1999, Wu 2009, Cai et al. 1996), results to be a suitable enzyme for PLA biodegradation able

to decrease the polymer molecular weight by hydrolysis accelerated in compost conditions.

PLA hydrolysis is a chemical reaction that depends on the pH and temperature conditions.

The degradation occurs at the particles surface and the polymer inside particles is not

degraded by this enzyme. Since polymer powders with a smaller particle size have a higher

surface area, the degradation speed depends on the total surface area of the particles.

About 60% of PLA films was degraded after 14 days from initial weight (Pranamuda et al.

1997) by using Mycolatopsis strain HT-32, which is an actinomycete was the first isolated

PLA-degrading microorganism (Ikura et al. 1999, Nakamura et al. 2001, Sakai et al. 2001,

Teeraphatpornchai et al. 2003, Jarerat et al. 2003, Tomita et al. 2004, Pranamuda et al. 2001.

Actually, there is little information about bacteria degrading PLA, just two thermophiles and

one mesophile have been isolated occasionally until now. One of them, isolated as first PLA-

degrading bacterium, was identified as a themophile belonging to Brevibacillus sp. (formerly

Bacillus brevi) (Torres et al. 1996). This microorganism is able to degrade PLA films

isolated from 153 soil samples with an enrichment culture technique performed at

temperature of 6°C (Ikura et al. 1999).

PLA-degrading fungi screen activity was first conducted by Kim et al. 2003. After 14 fungal

strains was tested, they found only two strains - F. Moniliforme and P. Roqueforti could

Introduction

- 31 -

assimilate or partially soluble PLA derivatives, D,L-lactic acid and the racemic oligomers

(Mw=1,000). They observed that one strain of F. Moniliforme was able to grow on a poly

(lactic acid-co -glycolic acid) copolyester (Mw=150,000) film, but its ability to mineralize

the polymer was poor.

A recent overview about PLA biodegradation (Tokiwa et al. 2004) reports that lactic acid

bacteria (LAB) and some filamentous fungi are the chief microbial sources of lactic acid. The

organisms that predominantly yield the L isomer are Lactobacilli amylophilus, Lacto-bacilli

bavaricus, Lactobacilli casei, Lactobacilli maltaromicus, and Lactobacilli salivarius. Strains

such as Lactobacilli delbrueckii, Lactobacilli jensenii, and Lactobacilli acidophilus give the

Disomer or mixtures of both.

PLA film degradation can occur by using Saccharo-thrix waywayandensis (renamed as

Lentzea waywayandensis) (Jarerat et al. 2003) and by Kibdelosporangium aridum (Wu 2009)

and it is an evidence that PLA can be degraded markedly by adding gelatin into the culture

medium.

The thermal properties for PLA based composites loaded with green coconut fiber (GCF)

were evaluated (Fukushima et al. 2009) and the biodegradation tests in Burkholderia cepacia

BCRC 14253 compost, showed that PLA can be completely degraded by this bacterium.

The photodegradation process was investigated by Tsuji et al. 2006 for some materials made

of PLA by using gel permeation chromatography, differential scanning calorimetry, tensile

testing, and polarization optical microscopy methods.

UV penetration into the specimens had no significant reduction effect on its intensity

(irrespective of the chemical structure and the crystallinity of biodegradable polyesters).

Although PLLA chains are photodegradable even in the crystalline regions, their

photodegradability of amorphous regions is lower. The significant increase in weight-average

molecular weight (Mw)/number-average molecular weight (Mn) was observed for PLLA-A

films, even there was small decrease in Mn by UV irradiation. Anyway, most of PLLA films

tensile properties of PLLA remain constant during UV irradiation.

1.5.6. PLA Biodegradation Mechanism in Compost

Iovino et al. 2008 used standard test methods for evaluating a composite aerobic

biodegradation.

Changes at different incubation times, confirmed by thermal properties‘ and SEM

micrographs, showed that the formation of patterns and cracks on the material surface aged

in the compost is an evidence of a significant loss of structure.

Biodegradation is caused by biological activity, especially by enzymatic action, leading to a

significant change in the chemical structure of the exposed material and resulting in the

production of carbon dioxide, water, mineral salts (mineralization) and new microbial

cellular constituents (biomass). It can occur by using two different conditions: aerobic in the

presence of oxygen and anaerobic with no oxygen available as Grima et al. (2000) reported.

From this research, the most productive phase for PLA mineralization occurred from 12 to

50 day (d) incubation. In addition, the PLA degradation was about 55.5% of its theoretical

carbon dioxide production, with a carbon content of 54.6 wt % in PLA. So this polymer was

degraded more easily for the presence of water inside the fibres.

Heterochain polymers, particularly those containing oxygen and/or nitrogen atoms in the

main chain, are generally susceptible to hydrolysis. For hydrolysis to occur, the polymer

must contain hydrolytically unstable bonds, such as ester, amide, or urethane, and it must


- 32 -

show some degree of hydrophilicity. In addition, the hydrolysis rate of the polymer is

affected by properties such as molecular weight, glass transition temperature, and

crystallinity and also hydrolysis conditions such as pH, temperature, and the presence of

enzymes and microorganisms. The hydrolysis of PLA polymers has been widely studied both

in vivo and in vitro. Hydrolytic degradation of poly (DL- and L-lactic acids) proceeds by

random hydrolytic chain scission of the ester linkages, eventually producing the monomeric

lactic acid. The mechanism by which PLA polymer degrade depends on the biological

environment to which it is exposed. In mammalian bodies, PLA is initially degraded by

hydrolysis and its oligomers are metabolized or mineralized by cells and enzymes. Abiotic

hydrolysis is known to participate at the initial stage of microbial biodegradation of PLA in

nature. However, the degradation rate has been shown to increase in the compost

environment in the presence of an active microbial community compared to the abiotic

hydrolysis (Hakkarainen et al. 2000).

Teramoto et al. 2004 gave another explanation for PLA degradation that could imply the

improvement of the interface interaction between matrix and fibre due to the presence of

maleic anhydride, therefore inhibiting water or microorganism penetration within polymers.

The decrease in melting temperature could be attributed to the gradual reduction of

polymeric chains with an increasing exposure time in the composting environment.

In the enzymatic PLA degradation mechanism (Ohkita et al. 2006), a surface erosion rather

than a non-specific inner degradation for unchanged in molecular weight by enzymatic

degradation. The mechanical properties loss after degradation was confirmed.

In neutral or alkaline medium the observed degradation for lactic acid oligomers, may be

explained by intramolecular trans-esterification, also called back-biting (De Jong et al.

2001).

Nucleophilic attack of the hydroxyl end group on the second carbonyl group leads to the

formation of a stable six-membered ring as an intermediate. This reaction is base-catalyzed,

since a base can interact with the hydroxyl end group, thereby increasing the nucleophilicity

of the oxygen atom (Fig. 1.11).

As evidence for the participation of the end group in the degradation, the hydroxyl end group

of lactic acid oligomer was modified by reaction with succinic anhydride to yield lactic acid

oligomer with a carboxylic acid end group. The degradation samples at pH 7 were analyzed

with MS and compared with the mass spectra of the degradation sample of unmodified lactic

acid oligomers at the same time points. In the studied time frame of 15 days, no significant

degradation of the lact-SA was observed, whereas the unmodified lactic acid oligomer was

almost completely degraded, showing the importance of the hydroxyl end group of the lactic

acid oligomer for hydrolytic degradation in alkaline medium.

Introduction

- 33 -

Figure 1.11. PLA Mechanism of Hydrolysis in Alkaline Environment (A) and Acid

Environment (B).

The preferential cleavage of the ester bond at the terminal hydroxyl end of the oligomer at

low pH can be explained as follows: the degradation is initiated by protonation of the OH

end group, followed by formation of an intra-molecular hydrogen bridge. Of all possible

intermediate structures, the five-membered ring is the most stable one (Fig. 1.10B). The

electrophilicity of the carbonyl group increased by hydrogen bridge formation and attack of a

water molecule at that site is therefore preferred. Lactic acid will be split off leaving lactic

acid oligomer with a DP of one unit less than the starting compound.

So under both acidic and basic conditions, the different ester groups in lactic acid oligomers

do not have the same susceptibility for hydrolysis. This means that the hydrolysis of these

oligomers does not proceed randomly as suggested for PLA (and related polymers such as


- 34 -

PLGA) but that, starting from the terminal hydroxyl group of the oligomer, the first and the

second ester bond are preferentially cleaved under acidic and basic conditions, respectively.

The degradation of bioerodable systems (implants, microspheres) based on PLLA and

PLGA, needs two remarks. First, due to the low concentration of the OH-end group in the

initial degradation phase of these systems, it is not unlikely that initially the hydrolysis

occurs randomly in the polymer chain (Arena et al. 2011). However, when the degradation

proceeds, the number of end groups increases, whereby the OH-end group-catalyzed

hydrolysis, as suggested here, might have an increasing contribution to the overall

degradation occurring in bioerodable systems.

Second, it has been demonstrated that within the PLLA and PLGA matrices the pH decreases

with increasing degradation. This is ascribed to the increasing number of COOH groups as

well as to the accumulation of water soluble, low molecular weight degradation products

inside these matrices.

Obviously, the fact that PLLA and PLGA matrices fully degrade when placed in an aqueous

environment suggests that the increase in hydrophilicity contributes more to the overall

degradation process than the decrease in pH.

1.5.7. The Role of the Organic Filler on the Biodegradation Behavior and Final Properties

of PHAs Composites.

Although biodegradable polymers have good biodegradability and biocompatibility, their

mechanical and thermal properties as thermoplastic elastomer, such as shock absorbance,

impact strength, tensile strength and thermal resistance, remain inadequate. Therefore, there

has been much interest in modifying their properties by the synthesis and/or modification of

polymer structure (Todo et al. 2007) that are time-consumable and/or uneconomical for the

production of plastic items made by these novel biodegradable polymers.

A solution suitable to improve the polymer biodegradation propensity and the material

mechanical properties is constituted by the blending with organic fillers. Depending on the

property requirement required for different applications, PHAs can be either blended, surface

modified or grafted with other polymers; enzymes or inorganic materials with the aim to

improve their mechanical properties and their biodegradation propensity (Doy et al. 1995,

Rezwan et al. 2006).

Biodegradable polymers including poly-saccharides and proteins can be inserted in

polymeric matrices as reinforcement. The same role is played by a variety of bio-fibers such

as lignocellulosic ones which are described in many research articles and reviews (Reis et al.

1996, Mohanty et al. 2000, Jandas et al. 2012).

Advantages of biofibres over traditional reinforcing materials such as glass fibres, talc and

mica are low cost, low density, high toughness, acceptable specific strength properties,

reduced tool wear, reduced dermal and respiratory irritation, good thermal properties, ease of

separation, enhanced energy recovery and biodegradability. The main drawback is their

hydrophilic nature which lowers the compatibility with hydrophobic polymeric matrix during

composite fabrications. The other disadvantage is the relatively low processing temperature

required due to the possibility of fibre degradation and/or the possibility of volatile emissions

that could affect composite properties. The processing temperatures for most of the biofibres

Introduction

- 35 -

are thus limited to about 200°C, although it is possible to use higher temperatures for short

periods (Mohanty et al. 2000).

Many composite materials are difficult to remove after the end of life time, as the

components are closely interconnected, relatively stable and therefore difficult to separate

and recycle. In the modern polymer technology it is a great demand that every material

should especially be adapted to the environment.

By embedding natural reinforcing fibres, (flax, hemp, ramie) into biopolymeric matrix made

of derivatives from cellulose, starch, lactic acid, etc; new fibre reinforced materials called

biocomposites were created and are still being developed (Keller et al. 2003).

Biocomposites consist of biodegradable polymers as continuous matrix and usually biofibres

as reinforcing element. Since both components are biodegradable, the composite as the

integral part is also expected to be biodegradable. Biofibres, i. e., natural polymers, are

generally biodegradable but they do not possess the necessary thermal and mechanical

properties desirable for engineering plastics. On the other hand, the best engineered plastics

are obtained from synthetic polymers, but they are non-biodegradable. A lot of R&D work

has been carried out on biofibre reinforced synthetic polymers. The composites of natural

fibres and non-biodegradable synthetic polymers may offer a new class of materials but are

not completely biodegradable.

The biofibres derived from annually renewable resources, as reinforcing fibres in both

thermoplastic and thermoset matrix composites provide positive environmental benefits with

respect to ultimate disposability and raw material utilization (Mohanty et al. 2000).

1.5.7.1. PHAs-Cellulose Derivatives Blends and Composites

Bourban et al. 1997 developed a biodegradable composite consisting of a degradable

continuous cellulosic fiber and a degradable matrix PHBV (HV19% mol) obtained by

impregnating the cellulosic fibers on-line with PHB/V powder in a fluidization chamber.

PHB/V-regenerated cellulosic fibers composite showed promising mechanical properties,

thus the cellulosic fibers significantly increased the stiffness and strength of the PHB/V

matrix while maintaining the biodegradability of this material.

Most of the natural fibers consist of single fiber packages, so called ‗fiber bundles‘ where the

cellulose is embedded in lignin which functions as a kind of matrix. Therefore, a natural fiber

(fiber bundle) can be considered as a naturally occurring composite.

Furthermore, the fiber properties differ depending on the fiber geometry. Moreover, due to

the varying chemical composition of natural fibers (cellulose, hemicellulose, lignin, pectin),

the orientation of the cellulose and the different structural defects along the fiber or the

fiber/matrix inter-phase, decrease consequently the composite mechanical properties

(Bledzki et al. 2010).

The higher aspect ratio (fiber length/fiber diameter; L/D) of the fine cellulose fibers may be

also the cause for a greater strength in the composite. For that reason, the stiffness of bio-

composites with man-made cellulose fiber is partially deteriorated compared to composites

with natural fibers that show a higher strength (Bledzki et al. 2010).

The incorporation of fibrous reinforcement causes a decrease in polymer chains mobility, as

shown by DMA analysis. PLA-based composites storage modulus remained much higher

than of un-reinforced PLA. The glass transition temperature derived from loss modulus


- 36 -

showed a slight shift to higher temperatures, if compared to matrix polymer. SEM

photographs showed fiber pull-outs from the polymer matrix: this is due to the surface

roughness, which is very smooth with regenerated cellulose. The adhesion was not strong in

any of the composites. The fiber orientation was analyzed via optical microscopy (Bledzki et

al. 2009).

Polylactic acid (PLA) nanocomposites were prepared using Cellulose NanoWhiskers

(CNWS) as a reinforcing element in order to assess the role of this filler in reducing the gas

and vapour permeability of the bio-polyester matrix. The crystallinity value and the fiber

dispersion in the polymer matrix, are connected with the CNWS treatment (solution casting,

acid hydrolysis, freeze-drying) as showed by TEM results.

From the DSC analysis, melting point and crystallinity increased with increasing CNWS

loading hence suggesting a filler-induced crystallinity development. The heat of fusion

seemed to increase slightly in the highly amorphous PLA bio-composites (Sanchez-Garcia et

al. 2008). From TGA results, it was concluded that the addition of low fractions of CNWS in

PLA matrix did not alter the matrix thermal degradation.

PLA nano-composites water permeability decreased with the freeze drying CNW addition by

up to 82% and the oxygen permeability by up to 90%: an optimum barrier enhancement was

found for composites containing loadings of CNWS below 3 wt% (Sanchez-Garcia et al

2010a).

The lowest water permeability was probably due to the high nano-dispersion of the

nanowhiskers across the matrix and the high crystallinity, the good interfacial adhesion and

dispersed morphology was seen in these nanobiocomposites (Sanchez-Garcia et al 2010a,

Sanchez-Garcia et al 2010b). The mechanical performance was evaluated lower than that of

neat PLA: this observation was ascribed to both the filler ranged screened being below the

percolation threshold and most importantly to filler-induced plasticization by sorbed

moisture. So CNWS exhibit novel significant potential in coatings, membranes and food

agro-based packaging applications (Sanchez-Garcia et al 2010a).

The plasticizing and compatibilizing action for nano-structured cellulose fibres and their

esters (Takatani et al. 2008), was demonstrated by the mechanical properties improvement,

when the fibres were added to the polymer in a concentration of 5% by weight. The

nanocomposite showed better barrier properties than pure PLA, being minor the free volume

that can be occupied by adsorbed water molecole, becouse during nucleation, the formation

of crystals (Sanchez-Garcia 2010).

1.5.7.2. PHA-Natural Fiber Composites

Many researchers (Zini et al. 2007, Shanks et al. 2004) investigated composites consisting of

a bacterial co-polyesters such as PHBHHx, and PHBV with 5% and 12% mol HV loaded

with flax fibers. A satisfactory reinforcing effect was observed in composites prepared by

compression molding and using long fiber mats (long fiber composites, LFC). The long flax

fibers successfully reinforced the bacterial PHBHHx copolyester. Surface acetylation or a

silane coupling agent employment is suitable for promoting fiber–matrix adhesion, thus

improving the mechanical properties of short fiber composites. The crystallization kinetics of

Introduction

- 37 -

PHBHHx is accelerated in the composite by the presence of fibers. The melting temperature

was influenced by the promoted adhesion and copolymerization. The bending modulus was

increased in the composites and DMA provided storage modulus of as much as 4 GPa at

25oC, with a smaller component as the loss modulus.

An earlier study showed that flax and P (3HB) had good interfacial adhesion, which was

decreased when plasticizers were present (Wong et al. 2002). Some plasticizer migrated from

the flax to the PHB and caused complex changes in the glass transition, crystallization and

crystallinity of the PHB.

The thermal and mechanical behavior of biotechnological polyester PHBV reinforced with

wheat straw fibers were reported by Avella et al. 2000. In order to improve the chemico-

physical interactions between the components, the reinforcing agent has previously been

submitted to treatment with high temperature steam, leading to fibers richer in cellulose and

more reactive.

The addition of straw fibers has been found to increase the rate of PHBV crystallization,

while it does not affect the crystallinity content. Furthermore, the wheat straw/PHBV based

composites showed higher Young's modulus and lower values of both stress and strain at

break than the neat PHBV matrix.

It was observed that the presence of straw did not affect the biodegradation rate evaluated in

liquid environments and in long term soil burial tests. In the composting simulation test, the

rate of biodegradation is reduced for composites with more than 10% straw content. The

morphology of the composites has also been investigated and correlated to the

biodegradation process.

Lignocellulosic fractions from wheat straw as natural fillers in loading composites of a

biodegradable polyester (poly(butylene adipate-co-terephthalate) (Le Digabel et al. 2004)

showing a reinforcing effect of these residues.

Bio-composites based on jute fibers and blends of plasticized starch and PHB were

investigated by Belhassen et al. 2009. Different amounts of glycerol and aliphatic polyesters

PHB have been added to native starch to obtain a processable biodegradable matrix. In the

same way natural jute fibers up to 30 wt.% loading were added to improve the mechanical

and thermal stability of the material. Significant enhancement in the mechanical properties

and water sensitivity were noticed by the addition of 8 wt % PHB.

The addition of jute strands to the starch-based polymer matrix leaded to a material with

higher capacities to support stresses keeping them even after 1.000 h of exposition under

humid environment. The addition of the fibers up to 30 wt. % content brings about an

enhancement in the strength and modulus of the material as much higher as the fibers loading

is important. This decrease in water sensibility and improvement in mechanical resistance

leads to a useful material in the field of packaging with the capacity of biodegradation after

working life.

PLA is a crystalline biodegradable polymer that has poor processing properties such as poor

impact strength, low heat distortion temperature, poor processability and relatively high cost

(Li et al. 2010). Copolymers of L-lactic acid and glycolic acid (PLGA) are frequently used in

biomedical applications to enable tailoring of flexibility and degradation rate.


- 38 -

A study on the PLA blending with soy flour (SF), wood flour (WF) and sodium bisulfite-

modified SF that was used for improving the PLA adhesion, is present in literature. The

coupling agent was methylenediphenyl diisocyanate (MDI) (0.5 wt. %). Mechanical and

thermal properties, morphology and relaxation characteristics of the blends were investigated

and the correlated results showed that MDI was an effective coupling agent for the WF/PLA

system in improving tensile strength and elongation. DSC results indicated that SF and

modified SF acted as nucleation agents and facilitated the PLA crystallization behaviour by

increasing the crystallinity percentage. Mechanical relaxation behaviors were well

characterized on the basis of DMA results. PLA and its blends exhibited highly

homogeneous relaxational dynamics in their transition from glass to liquid during heating

because of the high degree of hydrophobicity and densification effects of PLA, SF and MSF

(Li et al. 2010).

PLA biocomposites with abaca fibres were processed by using combined moulding

technology that were two-step extrusion coating process and consecutively injection

moulding.

The abaca fibres (30 wt. %) reinforcement effect enhanced E-Modulus, tensile strength and

the Charpy A-notch impact resistance for PLA/abaca based composites.

Abaca fibre causes in general better stiffness than man-made cellulose, while in comparison

to un-reinforced polymer; this natural material improves mechanical characteristics. Also the

physical bonding to the matrix, due to fibre surface roughness, seemed to have improvements

by using abaca fibres instead of manmade cellulose ones. Reinforcing PLA with natural

fibres (jute and abaca) led to better chemical bonding on the inter-phase as for PP composites

without coupling agent and the desired mechanical performances were obtained and analysed

by using tensile and impact tests.

The reinforcement promoted by fibres increased the tensile stiffness and strength

significantly; however, depending on the fibre type, different improvements of the

mechanical parameters were achieved. Within the natural fibre group, jute seems to achieve

the highest improvement (up to 30% in PLA composites). The obvious reinforcing effect is

caused by the fibre performance. SEM was carried out to study the fibre–matrix inter-phasial

adhesion. The fibre-size distribution was determined using optical microscopy and the

obtained results were compared to composites on PP basis with the same reinforcing fibres.

The broadest fibre-size distribution can be found in abaca composites.

Considering the fibre bundle diameter, the abaca fibre is characterised by a higher average

value compared to jute one. An increasing in the fibre diameter affects the aspect ratio and,

thus, decreases the mechanical performance of the composites. Jute fibre bundles undergoes

to separation during processing, allowing a better distribution and a favourable diameter,

which can be seen in the results of fibre-size distribution. Moreover, the critical fibre length

of abaca is much higher and the aspect ratio (LD) lower than those of jute and man-made

cellulose. Amongst others, this is the reason for deteriorated mechanical parameters of abaca

composites (Bledzki et al. 2010).

PLA/cotton composites can be reinforced by using lignin that improved the cotton fibres and

PLA adhesion: the relative composites can be compared with PLA/kenaf ones from the

mechanical properties that showed an increase for tensile strength and Young‘s modulus and

Introduction

- 39 -

a decrease for the impact resistance (Nishino et al. 2003). Lignin has also been added to PLA

in order to reduce its flammability, giving performance competitive with commercial

formulations (Reti et al. 2008).

PLA can also be used as a matrix material in composites with natural fibres such as flax as

reinforcement and triacetin as plasticizer for improving neat PLA and PLA/flax based

composites impact properties, rigidity and heat resistance (Oksman et al. 2003, Bax et al.

2008).

A decrease in tensile strength with the increasing of the triacetin content was observed

expecially in PLA/flax composites. The triacetin addition showed a positive effect on the

elongation at break values for neat PLA and PLA/flax based composites. The highest

triacetin addition (15%) clearly showed a negative effect for PLA/flax based composites,

strongly decreasing their stress and stiffness values.

The microscopy study of the composite microstructure showed poor adhesion between the

fibres and PLA matrix. The flax fibres were well dispersed in the PLA matrix and they are

separated in single fibres due to the compounding process. PLA thermal properties, which

are a drawback for PLA, were improved with an addition of flax fibres (Oksman et al. 2003).

Agricultural co-products of the alternative oilseed crops, cuphea (C), lesquerella (L) and

milkweed (MPLA and various levels of co-product (0–45%, w/w) derived from alternative

oilseed crops, were compounded by twin-screw extrusion and injection molded with PLA

polymer for adjusting the desired mechanical properties. As co-product content increased,

tensile strength for all PLA composites decreases. PLA-C exhibited increased stiffness, while

the modulus of PLA-M and PLA-L decreased slightly. So, PLA-M showed extensive stress-

cracking under tensile stress and exhibited an elongation value 50–200% greater than the

PLA control. Acoustic emission showed ductile behavior of the PLA-M composite

(Finkenstadt et al. 2007).

Some other works (Petinakis et al. 2010, Yu et al. 2006, Wang et al. 2007) were carried out

with the aim to test the effect of hydrophilic fillers (starch and wood-flour) on the

degradation and the decomposition of PLA based materials was investigated. Both starch and

wood-flour accelerated thermal decomposition of PLA starch exhibited a greater impact than

wood-flour. The PLA decomposition temperature was decreased from about 397 °C to 352

°C when the starch content was increased to 40% by weight. In the case of the wood-flour

component, the decomposition peak of PLA decreased to about 362 °C. Small polar

molecules may have been produced during decomposition of starch and wood-flour (Yu et

al. 2006).

The wood flour use in PLA based composite materials makes better their tensile strength and

elongation at break values, whereas the wood soy plays a role of nucleant agent, facilitating

PLA crystallization and increasing its percentage (Li et al. 2010, Perego et al. 1996).

The wood flour eliminates PLA isothermal crystallization, thus its fibres being characterizing

by major fibres dimensions than soy ones, PLA crystallites formation resulted inhibited. The

chains mobility makes difficult the crystals growth (Li et al. 2010).

Biobased and biodegradable polylactide (PLA)-pine wood flour (PWF) based composites

were investigated (Pilla et al. 2010) as a means to reduce the overall material cost and tailor

the material properties. The tensile modulus of the PLA-PWF composites increased with the

PWF content whereas the toughness and strain-at-break values decreased. The tensile


- 40 -

strength remained the same irrespective of the PWF content (up to 40%), while the degree of

cristallinity showed a significant increment.

1.5.7.3. PHAs Lignin Blends and Composites

Lignin is primarily a structural material to add strength and rigidity to cell walls and it

constitutes between 15 wt% and 40 wt% of the dry matter of woody plants. Lignin is more

resistant to most forms of biological attack than cellulose and other structural

polysaccharides. In vitro, lignin and lignin extracts have been shown to have antimicrobial

and antifungal activity, it acts as antioxidant, it absorbs UV radiation, and it exhibits flame-

retardant properties.

Lignin is a cross-linked macromolecular material based on a phenylpropanoid monomer

structure. Typical molecular masses of isolated lignin are in the range of 1000–20,000 g/mol,

but the degree of polymerization in nature is difficult to measure, since lignin is invariably

fragmented during extraction and it consists of several types of substructures which repeat in

an apparently random manner. Blending of PHA with lignin is one possible strategy for

overcoming the mechanical disadvantages of PHA.

Thermoplastic blends of several biodegradable polymers with organosolv lignin and

organosolv lignin ester based on both solvent casting and melt processing, were investigated

by Ghosh et al. 2000.

An addition of up to 20 wt% lignin to PHB gives improvements for Tg, melting point,

Young‘s modulus, and the degree of crystallinity. The addition of lignin reduced the

crystallinity of PHB more than addition of lignin butyrate, suggesting greater compatibility

of PHB with lignin than lignin butyrate.

The effect of 1 wt% lignin on the nucleation of PHB by studying the kinetics of both

isothermal and nonisothermal P(3HB) crystallization, was investigated by Weihua et al.

2004. DSC results showed that not only lignin act as a nucleating agent, decreasing the

activation energy of crystallization, but it reduced the size of the spherulites to give a less

brittle material.

The nucleant agents addition permits the control of crystallization rate for PLA, so its final

cristallinity and the optimization for the processing conditions in the injection process can

reached better mechanical properties that reflect an increment for tensile strength and

material module (Harris 2008, Yu et al. 2012).

The miscibility, between PHB and bagasse soda lignins (having distinct chemical group

functionality) based on the Tg of their blends, was examined by Mousavioun et al. 2010.

The Tg values were higher for the blends obtained from the lignin fraction with an higher

content of xylan and phenolic hydroxyl groups, but lower methoxyl and carboxylic acid

content, implying that the association between lignin and PHB is probably related to the

chemical functionality of the lignin polymer as the molecular weights of these lignin

fractions are similar, approximately 2400 g/mol. In fact it was shown by FTIR that the

miscibility between PHB and soda lignin was due to hydrogen bond formation between the

carbonyl group of PHB and the phenol hydroxyl group of lignin.

The major drawback connected with PLA applications, is constitued by its high glass

transition temperature (Tg= 60°C) that gives poor processing properties, easy formation of

cracks in based PLA composite materials (Li et al. 2003), low strain at break and high

Introduction

- 41 -

module values (Pillin et al 2006), that are all restrictive features for the PLA use in the rigid

and thermoformed packaging industry.

A strategy employed to decrease the material rigidity is represented by the blending with

organic (lignin) and inorganic fillers or natural fibres such as cotton, wood, Kenaf (Li et al.

2003, Graupner 2008, Li et al. 2010, Bledzki et al. 2009, Bledzki et al. 2010, Sanchez-Garcia

2010, Finkenstadt 2010, Pluta et al. 2002).

The lignin loading in a percentage below 20%, can decrease the finite product price, giving

the biodegradability requisite to thermoplastic polymers (Li et al. 2003).

The lignin shows also an adhesive and strentghing action between ligno-cellulosic fibres

such as kenaf (10-14% of lignin) and the polymeric matrix PLA, as observed from the

increment of the tensile strength (52.88 MPa) for the PLA/Kenaf composite (Graupner

2008), where the structure becomes a multilayer created by the linkages between its inner

layers.

The thermal and mechanical properties of the biodegradable blends of PLLA and lignin

prepared by manual mixing have been investigated. Both the results of DSC and FTIR

spectral analyses indicated the existence of intermolecular interaction between PLLA and

lignin.

Furthermore, FTIR elucidated the existence of an intermolecular hydrogen-bonding

interaction.

The mechanical properties of PLLA/lignin blends showed that the maximum strength and the

elongation at break values for the material decreased whereas the Young‘s modulus

remained almost constant when the lignin content reached 20%. Thermogravimetrical

analysis showed that lignin would accelerate the thermal degradation of PLLA when the

content of lignin was more than 20%. Compared with some other biodegradable polymers,

the blend of PLLA and lignin is thought to be a promising material, because the material

properties, as well as the price, are at an acceptable level when the content of lignin is less

than about 20% (Li et al. 2003).

1.5.8 The Role of the Organic Filler on the Biodegradation Behavior and Final Properties

of Petro-based Polymers Composites (PE, PP, PS)

Biofibers, natural lignocellulosics, have an outstanding potential as reinforcement in

thermoplastics. The preparation of lignocellulosic composites with a good interfacial

adhesion is generated by a combination of fiber and matrix modification and methods. PP

matrix was modified by reacting with maleic anhydride and subsequently bonded to the

surface of the modified lignocellulosic component, in-situ. A silane agent was used as a

surface modifier and the modified fibers are then extruded with the modified polymer matrix

to form the compatibilized composite. Various reactions between the lignocellulosic

fiber/filler and modified polymer chains were performed by Karnani et al. 1997 with the aim

to improve the interfacial adhesion by using the formation of covalent bonds between the

fiber surface and matrix. These composite blends were then injection molded for mechanical

characterization and the results were reported (Karnani et al. 1997).

Composites made by blending ethylene vinyl acetate (EVA) with wood fiber (WF)

(uncompatibilized) and EVA with poly (ethylene-co-glycidyl methacrylate) (EGMA)-WF

(compatibilized), were investigated for the morphology as well as mechanical, thermal, O2

permeability, and water absorption properties.


- 42 -

The results showed that there is a weak interaction between EVA and WF able to confere

poor properties to the obtained composites. The presence of a compatibilizer such as EGMA

improved

the mechanical properties with the increase of EGMA content, whereas thermal stability,

oxygen permeability and water absorption showed a decrement. The compatibilizer becomes

chemically bonded to the hydrophyllic WF, and it makes easier the wetting of the

hydrophobic polymer chain. (Dikobe et al.2007).

Natural fibers, used as fillers and reinforcement for polymers, represent one of the fastest-

growing types of polymer additives (Czvikovszky et al. 1996, Bengtsson et al. 2005). In

some studies, biobased composites are formulated from a blend of natural fibers, including

cane fibers, bamboo, oat, kenaf, hemp, flax, jute and sisal, wood fiber (WF), and

thermoplastic polymers, such as polypropylene and polyesters.

The obtained composites resulted stronger, stiffer, and light-weight, because the natural filler

plays a role of reinforcement agent for the matrix.

The structure-property relationship between wood flake- high density polyethylene (HDPE)

based composites was investigated in relation to the matrix agent melt flow behavior and

processing technique (Balasuriya et al. 2001). The flake distribution and flake wetting were

optimized with the aim to obtain better mechanical properties, as evidenced by impact

strength values.

The polymer matrix provides an environment for the fibers and it protects them from

scratches that might cause them to fracture under low stress. The fibrous filler adds strength

to the more fragile polymer material by shouldering much of the stress that was transferred

from the polymer to the fiber through their strong interfacial bonds (Balasuriya et al. 2001).

Different particle sizes can be used to produce composites with different properties

depending on their distribution. The resulting WF–polymer composites have high specific

mechanical properties (Selke et al. 2004).

The mechanical properties of compression-moulded polystyrenes loaded with sawdust wood

residue of softwood and hardwood species, with different particle size distribution, were

investigated by Maldas et al. 1988. The tensile modulus at 0.1% strain and the tensile

strength, elongation and energy at the yield point are reported. Moreover, to improve the

compatibility of the wood fibres with the polymer matrices, different treatments (e.g. graft

copolymerization) and coupling agents (e.g. silanes and isocyanates at various

concentrations) were also used. The extent of the improvement in mechanical properties

depends on the fibre loading, on the particle size of the fiber, on the concentration and

chemical structure of the coupling agents, and on whether special treatments (e.g. coating or

grafting of the fibre). The mechanical properties of the composites are improved up to 30%

in the case of fibres having a mesh size 60 and when up to 3% of isocyanates was used

(Maldas et al. 1988).

Different chemical substances have been used as compatibilizers of wood polymer

composites by various researchers.

To improve the compatibility between the WF and the LLDPE matrix, Liao et al. 1997

treated the WF with titanate coupling agents or grafted it with acrylonitrile. Both treatments

Introduction

- 43 -

resulted in an improvement of the mechanical properties for the composites compared with

the same ones loaded with the untreated WF.

The same results were also observed for the WF-PS based composites loaded with coupling

agents (e.g., silanes and isocyanates at various concentrations) that showed a compatibilizing

effect.

Another drawback is the high moisture absorption of the WFs. Moisture absorption can lead

to swelling of the fibers, and concerns on the stability of the fiber composites cannot be

ignored (Tserki et al. 2005). The moisture absorption of the fibers can be minimized by the

encapsulation into the polymer or by adopting chemical modification of some hydroxyl

groups present in the fiber. This second option reflects an increase in the cost of the fiber.

Good fiber-matrix bonding can also decrease the rate and amount of water absorbed by the

composite.

Fibres from pine or eucalyptus wood and also one-year crops such as coir, sisal, etc. are all

good candidates for replacing glass fibres in PP based composites (Espert et al. 2004) even if

the poor resistance towards water absorption is one of the drawbacks of these natural

composites materials.

A decrease in tensile properties, such as lower values of Young‘s modulus and stress at

maximum load was demonstrated, showing a great loss in mechanical properties of the

water-saturated samples compared to the dry samples. The morphology change was

monitored by scanning electron microscopy analysis before and after exposure to water and

the devastating effect of water on the fibre structure was shown.

Espert et al (2004) reported that WF-PP composites, without compatibilizer, show

mechanical properties dramatically affected by water absorption.

The ethylene vinyl acetate (EVA) acts a role of compatibilizer, decreasing the adsorbed

water amount (Espert et al. 2004): the adhesion to the PP matrix, and vinyl acetate could

bond be connected to its acetate groups to the hydroxyl groups on the fibres.

Elvy et al. 1995 reported that the usage of coupling agents such as vinyltriacetoxysilane,

before impregnation with MMA in order to evaluate the effects of the silane coupling agent

on the physical properties of wood-polymer composites. A decreasing for the strand between

the polymer and the cell wall of the wood connected with a reduction in water absorption

were reported.

Very little has been reported on EVA-natural fiber composites. Malunka et al (2006)

investigated EVA–sisal fiber composites in the absence and presence of dicumyl peroxide

(DCP). This oxo-polyethylene is able to initiate crosslinking, being effective in grafting EVA

to sisal fiber and creating composites with better physical properties.

Sedlakova et al 2001 analyzed the role of poly (ethylene-co-methacrylic acid) copolymer

(EMAA) as compatibilizer loaded into low-density polyethylene (LDPE) and wood flour

based composites. Both mechanical and dynamic mechanical properties showed that EMAA

is able to promote better interactions between LDPE and WF. A high content of WF and low

content of EMAA in the system yield materials with high modulus and high tensile strength.

Polymer blending is an effective way to obtain materials with specific properties. Most

polymers are immiscible, therefore, blending them, it is possible to obtain heterogeneous

morphologies.


- 44 -

Immiscible blends have quite lower mechanical properties than those of their components, so

methods to improve adhesion between two immiscible blends, have been a subject of

considerable research activities (Xanthos et al. 1991). When the copolymers may be added

separately or formed in situ by reactive processing, it is supposed that the chemical reaction

between the components inside the blend, may cause an interfacial tension able to decrease

the adhesion force at the interface. A variety of reactive copolymers have been identified as

effective compatibilizers in earlier literatures (Xanthos et al. 1991).

Natural organic fillers such as wood fiber have traditionally composed a small percentage of

the filler market for thermoplastics when compared to mineral or glass-fiber reinforcements.

These fibres (Ton-That et al. 2009) offer many advantages such as (i) low density, (ii) high

strength and modulus values, (iii) renewable nature, and (iv) less breakdown of the fiber

during mixing than the commonly used inorganic reinforced fibers. The main problem that

occurs by using the wood fiber – thermoplastic composites is constituted by the poor

interfacial adhesion between the hydrophobic polymer matrix and the hydrophilic wood

fiber. This can be solved by adopting chemical modifications on the wood fiber surface, e.g.,

grafting of a short-chain molecule onto the wood fiber surface as well as using various

coupling agents / adhesion-promoting agents such as stearic acid and maleated propylene

wax. Moreover, the method of preparation for the composites also affects the ultimate

properties of these latter.

From the previous work, the physical and mechanical properties of wood may be improved

by preparing composites of wood with vinyl monomers. However, since most vinyl

monomers are non-polar, there is a little interaction between them and the hydroxyl groups

of the cellulose fibres. Therefore the components inside the wood polymer composite (WPC)

simply bulks the wood structure by filling the capillaries, vessels and other void spaces

within the wood. It can be deduced that if the bonding were placed between the impregnated

polymers and the hydroxyl groups on the cellulose fibers, the physical properties of WPC

may be further improved (Elvy et al. 1995) .

The interfacial adhesion between the wood fiber treated with two different titanate coupling

agents and grafted acrylonitrile loaded into LLDPE matrix, was investigated (Liao et al.

1997).

The results showed a higher ratio of grafting for the untreated fibers compared to the pure

ones, as observed from SEM analysis.

LLDPE loaded with the untreated wood fibers showed more fibers pullout from the matrix

during the fracture, whereas the grafting modification made the fiber easily deformable and it

enhanced fiber adhesion at the interface. The failure of the material was caused by fiber

fracture rather than by fiber pullout from the matrix.

The wood fibers grafted with acrylonitrile or filled with a titanate based agent were loaded

into LLDPE matrix. The tensile strength and elongation at break values, recorded for the

treated composites, showed an increment with the increasing in the grafting ratio for the

wood fiber. The modulus was not much affected by the fiber treatment and it was dependent

primarily on the wood fiber concentration in the material.

Introduction

- 45 -


Properties of PHAs

Some recent developments in the nano-biocomposites area for bio-based packaging

constitute the potential for the nano-fillers in the food packaging sector. The addition of

different types of clays, bio-fibers and their nanowhiskers, carbon nanotubes and carbon

nano-fibers to biopolyester matrices (PLA, PCL, PHB and PHBV) led not only to barrier

improvement but also to obtain novel functionalities such as UV and Visible protection and

to control the release of antimicrobial substances.

PHAs based composites mechanical properties can be improved also by adding epoxy chain

extenders, which maintain or increment the molecular weight at the polymer processing

temperatures, at which depolymeraization or chain scission reactions can occur, being

responsible of the viscosity and elasticity lack for the melt and of the material thermal and

mechanical properties (Pilla et al. 2009).

Lim et al. 2007 prepared hydrophobically modified silica hybrid composites based on

biodegradable PHBHHx using simple melt compounding and they investigated the effect of

hydrogen bonding on the crystallization behavior of the composites. The intermolecular

hydrogen bonding between PHBHHx and silica increased gradually with the increase of

silica content. The silica reduced the non-isothermal and isothermal crystallization rates of

PHBHHx in the hybrids, and these were ordered by the strength of intermolecular hydrogen

bonding between the PHBHHx and silica.

Recently, the utility of inorganic nanoparticles as additives to enhance the polymer

performance has been established. Various nanoreinforcements currently being developed

are nanoclay (layered silicates), cellulose nanowhiskers, ultra fine layered titanate, and

carbon nanotubes (CNs). Of particular interest are polymer and Organically Modified

Layered Silicate (OMLS) nanocomposites because of their demonstrated significant

enhancement, relative to an unmodified polymer resin, of a large number of physical

properties, including barrier, flammability resistance, thermal and environmental stability,

solvent uptake, and rate of biodegradability of biodegradable polymers (Ray et al. 2003). The

main reason for these improved properties in polymer/layered silicate nanocomposites is the

strong interfacial interactions between matrix and OMLS as opposed to conventional

composites (Ray et al. 1995, Chen et al. 2002 259, Chouzouri et al. 2007).

Maiti et al. 2003 reported the first preparation of PHB/OMLS nanocomposites (PHBCNs) by

melt intercalation method by using three different kinds of OMLS.

XRD patterns clearly showed the formation of well-ordered intercalated nanocomposites,

whereas TEM image of PHBCNs also supported the formation of the intercalated structure.

The fate of the polymer after nanocomposites preparation was measured by GPC. The

nanocomposites based on organically modified MMT showed severe degradation but

surprisingly no degradation was found with nanocomposites based on organically modified

fluoromica. There is no explanation how organically modified fluoromica played to protect

the system, but present authors believe presence of Al Lewis acid sites, which catalyze the

hydrolysis of ester linkages at high temperature, may be one of the reasons. The degradation

started just after one week and at the initial stage the weight loss was almost the same for

both PHB and its nanocomposites. Deviation was occurred after three weeks of exposure, but


- 46 -

degradation tendency of nanocomposites was suppressed. The intercalation of OMLS, inside

the nanocomposites, is able to make slow the biodegradation of PHB polymeric matrix and

to improve the barrier properties of the matrices.

PHB or PHBV clay nanocomposites can be prepared via solvent casting (Lim et al. 2003) or

by solution intercalation methods (Wang et al, 2005). FTIR analyses showed that two distinct

different phases coexisted. The thermal stability of the PHB/OMMT based nanocomposites

was also observed with the increasing of OMMT content. At higher clay contents (>6 wt%),

the nanocomposites degradation rates decreased due to restricted thermal motion of the

polymer chains in the OMMT interlayer. From both XRD and TGA analyses, the

aggregation tendency of OMMT was also found to increase with the OMMT content in these

systems and led to the intercalated structure.

The PHBV based nanocomposites showed a decrease in biodegradtion propensity in soil

suspension, melting temperature and enthalpy of fusion (DHm) increasing the OMMT

amount. It was demonstrated that the small-sized PHBV spherulites were formed, the

crystallinity of PHBV reduced and the range of the processing temperature for the

nanocomposites enlarged by intercalation of OMMT with PHBV. The biodegradability of

PHBV/OMMT based nanocomposites was related to the interaction and adhesion of PHBV

and OMMT, water permeability, degree of crystallinity, and anti-microbial property of

OMMT.

Hablot et al. 2008 reported that PHB enhanced degradation can also be caused by

decomposition products of clay organomodifiers which have a catalyzing effect on the

thermal or thermomechanical degradation. Eventually, the biodegradation studies also

highlighted the difference between montmorillonite and fluoromicas since the initial

degradation rate of PHB with MMT-NH3+(C18) was higher than with fluoromicas.

Choi et al. 2003 described the microstructure as well as the thermal and mechanical

properties of PHBV–organoclay (Cloisite 30B, a monotallow bis-hydroxyethyl ammonium-

modified montmorillonite clay) nanocomposites with a low clay content.

XRD and TEM analyses clearly confirmed that intercalated nanostructures were obtained,

dueing to the strong hydrogen bond interactions between PHBV and the hydroxyl groups of

the C30B organo-modifier.

The nanodispersed organoclay acted as a nucleating agent, increasing the temperature and

rate of PHBV crystallization. Moreover, the DSC thermograms revealed that the crystallite

size was reduced in the presence of nano-dispersed layers since the PHBV melting

temperature are shifted to lower temperatures. Thermogravimetric analyses revealed that the

temperature corresponding to 3% of weight loss increased with C30B content (+10◦C with 3

wt% of filler).

They explained these trends by the nanodispersion of the silicate layers into the matrix and

thus concluding that the well-dispersed and layered structure accounts for an efficient barrier

to the permeation of oxygen and combustion gas. Eventually, the mechanical properties

showed that clays can also act as an effective reinforcing agent since the Young‘s modulus

value significantly increases from 480 to more than 790 MPa due to strong hydrogen

bonding between PHBV and C30B.

Introduction

- 47 -

As clay organo-modifiers, also ammonium surfactants can be used, as suggested by Bordes

et al. 2009, which investigated the clay effect on thermal and thermo-mechanical degradation

of two PHBV grades and the neat PHB. The PHBV thermal degradation studies, with and

without quaternary ammonium cations, based on Tg data showed that all surfactants have an

effect on the polymer degradation. This effect is more pronounced when the initial Mw of

the polymer is low. The results regarding the thermal stability in the presence of surfactants

led to two additional statements: (i) the degradation mechanisms of both the purified PHBV

and the surfactant in the blend are interdependent, (ii) S-Alk and S-Bz appear to influence

PHBV thermal degradation the most. All these conclusions are totally in agreement with the

results obtained for PHB. The thermo-mechanical study has led to the same conclusions

regarding the effect of surfactants since the mechanical energy, and thus the viscosity, is

higher for neat PHBV. Moreover, according to the Mw values of PHA after processing, S-Bz

appears to be the most degrading surfactant. In addition to the molecular weight decrease, the

recorded low torque values could also be explained by a lubricating action of the surfactants

and/or of their degradation by-products.

PHBV processing with three-layered clay types using different mixing methods was

examined by Cabedo et al. 2009, with the aim to detect the effect of processing time, clay

type and content on polymer molecular weight and composite morphology. PHBV molecular

weight (Mw) decreased by 38% after extrusion processing and was further reduced in the

presence of montmorillonite (MMT). When PHBV was processed with kaolinite as additive,

no further reduction in polymer molecular weight was observed. Molecular weight decreased

as the MMT clay content increased from 1 to 5 wt %.

The results suggested that the release of tightly bound water from clay surfaces at high

temperatures may be responsible for PHBV degradation during processing. Evidence also

points to the possibility that the surface modifier present in organically modified MMT may

catalyze PHBV degradation in some way. X-ray diffraction studies indicated an intercalated

morphology in the presence of modified MMT but good dispersion was also achieved when

unmodified kaolinite was blended with PHBV. They concluded that the intercalated

morphology was found to be predominant when PHBV/MMT clay nanocomposites were

obtained either by solvent casting or melt mixing. Sub-micron aggregates were obtained

when the mixing method is the melt blending with unmodified kaolinite.

A nano-composite based on PLA and a layered silicate was prepared by adding trimethyl

octadecylammonium cation, an organically modified layered silicate (OMLS). WAXD and

TEM analyses confirmed that montmorillonite silicate layers were intercalated and nicely

distributed in the PLA-matrix. The neat PLA material properties improved remarkably after

nano-composite preparation. The neat PLA biodegradability and the corresponding nano-

composite one were studied under compost, and the rate of biodegradation of neat PLA

increased significantly after nano-composite preparation (Sorrentino et al. 2007, Sinha Ray et

al. 2002b).

Excellent yielding results for PLA/organically modified synthetic fluorine mica (OMSFM)

nano-composites, were obtained by adding synthetic fluorine mica (SFM), modified with N-

(coco alkyl)-N,N-[bis(2- hydroxyethyl)-N-methylammonium cation, that was nicely

distributed to a nanoscale level (Sanchez-Garcia et al. 2010a). All the nano-composites


- 48 -

exhibited dramatic improvement in various materials properties such as the rate of

crystallization, mechanical and flexural properties, heat distortion temperature, and O2 gas

permeability, with a simultaneous improvement in biodegradability for neat PLA. When

these composites are disposed of in compost, they can be safely decomposed into CO2 and

H2O through the activity of micro-organisms.

The barrier properties of PLA based nano-composites can also be improved by sinergistic

interactions between the superficial area of the inorganic filler layers and the polymer, that

try origin from intercalation and/or esfoliation of the inorganic structure (Pluta et al. 2002,

Chang et al. 2003, Kasuga et al. 2003).

Inorganic components such as talc or a complex of phosphoric ester combined with

hydrotalcite made better PLA melt crystallization, decreasing the material crystallization

temperature (207°C), thus the spherulites do not increase their dimensions (Urayama et

al.2003).

The gas barrier properties had been shown to improve dramatically upon exfoliation of clay

platelets in a number of polymeric matrixes. The mechanism for the improvement was

attributed to the increase in the tortuosity of the diffusive path for a penetrating molecule. In

agreement with the crystallinity data, which was found to generally increase with increasing

filler content, oxygen but specially water and D-limonene permeability coefficients were

seen to decrease to a significant extent in the bio-composites and an optimum property

balance was found for 5 wt % of clay loading in the biopolymer. Increasing clay content, the

light transmission of these biodegradable bio-composites decreased by up to 90% in the UV

wavelength region due to the specific UV blocking nature of the clay used (Sanchez-Garcia

et al. 2010a, Sinha Ray et al. 2003).

PLA/clay nano-composites loaded with 3 wt% of organo-modified montmorillonite and

PLA/clay micro-composites containing 3 wt% of sodium montmorillonite were prepared by

melt blending. A plasticizer like PEG 1000 (20 %wt.) was used (Yang et al. 2007, Paul et al.

2003).

The nano and micro-composites morphology and thermal properties were investigated

making a comparison with the unfilled PLA. As the polymer matrices of these samples could

be considered amorphous by their thermal histories (and, therefore, transparent), the opacity

of the Na+ montmorillonite-filled composite might have resulted from the light scattering on

the filler particles with their size comparable with the light wavelength. So the transparency

of the other composites suggested good dispersion of nano-clay particles within the polymer

matrices without noticeable agglomeration in areas of size comparable with the wavelength

of light.

The polymer chains intercalation between the alumino-silicates layers and morphological

structure of the filled PLAs were performed with DSC, TGA, XRD, SEC, and POM.

XRD measurements showed a good affinity between the organo-modified clay and the neat

PLA able to form intercalated structure in the nano-composite. The interlayer migration of

poly (ethylene glycol) 1000 plasticizer is mainly provocked by the polarity difference

between the PLA matrix and this type of clay.

DSC and LM analyses showed that the nature of the filler affected the ordering of the PLA

matrix at the molecular and super-molecular levels. According to TGA analysis, PLA based

Introduction

- 49 -

nano-composites exhibited improvement in their thermal stability in air. The significant

increase in thermal stability, even at very low levels of filler, for the nano-composite under

thermo-oxidative conditions suggested a change in the degradation mechanism.

At constant filler level, it appeared that among all the clays studied, the montmorillonite

organo-modified by bis-(2-hydroxyethyl)methyl (hydrogenated tallow alkyl) ammonium

cations brought the greater effect in terms of thermal stability. Increasing the amount of clay

allowed delaying the onset of thermal degradation of the plasticized polymer matrix by using

WAXS and DSC analyses. So a real competition between PEG 1000 and PLA for the

intercalation into the interlayer spacing of the clay exists (Chang et al.2003) as shown by the

morphological analyses (Paul et al. 2003).

A greater degree of disorder, which led to stronger interfacial strength and consequently

higher storage modulus for the organoclay based composites. It appeared that the crystal

network of the polymer contributed to the reinforcement that needed to amorphous

composites with desired properties controlled by the interfacial strength with dispersion of

clays (Chowdhury 2008).

For hybrid films, the tensile properties initially increased but then decreased with the

introduction of more of the inorganic phase. The hybrids ultimate strength increased

remarkably with the organoclay content and it possessed a maximum value for each clay

content loading.

Above the clay loadings corresponding to the ultimate strengths, all the hybrids strength

values started to decrease. Dispersion was very important because failure to disperse could

induce agglomerates. Failure initiated a lot of MMT agglomerates, and this meant that cracks

were initiated on and propagated inside them: a high MMT content led to an agglomeration

of MMT particles, which reduced the tensile strength. The modulus enhancement was

correlated to the resistance exerted by the clay itself and to the orientation and aspect ratio of

the clay layers. Additionally, the stretching resistance of the oriented backbone of the

polymer chain in the gallery also contributed to enhancing the modulus that started to

decrease when the organoclay content was greater than a critical weight percentage. PLA

elongation at break increased with the introduction of an organoclay in high content.

However, a certain organoclay loading corresponded to the maximum value, giving a 31–

41% increase in the elongation at break value. Therefore, it can be concluded that in a certain

organoclay content range, its introduction led to both a strengthening and a toughening for

PLA matrix. A further increase in the organoclay content led to a decrease in the elongation

at break, even though the elongations at break of nano-composites containing 8 wt %

organoclays were still much higher than those of neat PLA. From these results, it appeared

that there was an optimal amount of organoclay needed in a hybrid to achieve the greatest

improvements in its properties that might result from the strong interfacial interaction

between PLA and the organoclay (Chang et al. 2003).

Organophilic montmorillonite was obtained by the reaction of montmorillonite (MON) and

DiStearyldimethylAmmonium Chloride (DSAC). The modified clay and PLLA, were

solvent-cast blended using chloroform as co-solvent. The PLLA–clay blends were

investigated in regard of their structure and properties using thermal measurements that

revealed that cold crystallization took place in the solvent cast PLLA, and that the clay


- 50 -

served as a nucleating agent. From small and wide-angle x-ray scattering measurements, it

was found that silicate layers forming the clay could not be individually well dispersed in the

PLLA–clay blends. So the clay existed in the form of tactoids, which consist of several

stacked silicate monolayers. These structures formed a remarkable geometrical network in

the blend film surfaces. They can be layed almost parallel to the film surface, and they were

stacked with the insertion of PLLA crystalline lamella in the thickness direction of the film.

During the blend drawing process, fibrillation took place with the formation of plane-like

voids developed on the plane parallel to the film surface. Furthermore, delamination of the

silicate layers did not occur even under the application of a shearing force (Ogata et al.

1997).

PLA thermal degradation and its blends or composites have also been widely reported by

Bourban et al. 2007. They analyzed the filler dispersion in the polymer matrix and the

thermal degradation rate showing that the relative constants of the loaded nanocomposites

were significantly lower than those of the unloaded polymers and their micro-composites in

the air.

TGA data and kinetic analysis results also supported the findings that the thermal stability of

the amorphous PLA and its composites was higher than that of the semicrystalline polymer

and its composites and the thermal stability of the nano-composites was higher than that of

the micro-composites (Bourban et al. 1997).


Properties of Petro-based Polymers (PE, PP, PS)

Plastics occupy wide place in the applications of automotive, electronics and house goods.

Especially reinforced plastics become popular because of their high strength besides their

advantages of low weight and easy manufacturability (Altan et al. 2010).

Mechanical and morphological properties of Polypropylene (PP) and High Density

Polyethylene (HDPE) matrix composites reinforced with surface modified nano titan dioxide

(TiO2) particles were investigated. Surface modification was made by coating the nano

powders with Maleic Anhydride grafted Styrene Ethylene Butylene Styrene (SEBS-g-MA)

and silane, respectively.

After surface modification, they assessed thermal and morphological analysis for PP/TiO2

and HDPE/TiO2 based composites that were obtained by loading of 1 wt.%, 3 wt.% and 5

wt.% of the inorganic filler.

SEBS-g-MA provided bridging effect between TiO2 particles and polymer matrix while

silane played a role of dispersing agent. Depending on that, homogenous structures without

agglomeration were obtained. Reinforced HDPE and PP moldings gave higher tensile

strength and elastic modulus due to the rigid structure of TiO2.

Slight increment was seen in stress at break value, whereas elongation and impact strength

values decreased due to the stiffness of the nano titan dioxide (Altan et al. 2010).

The influence of filler size and content on the properties (thermal conductivity, impact

strength and tensile strength) of Al2O3/high density polyethylene (HDPE) based composites

were studied by Zhang et al. 2011. Thermal conductivity and tensile strength of the

composites increased decreasing the aluminium oxide particle size, whereas the dependence

of impact strength on this parameter is more complicated. SEM micrographs of the fracture

Introduction

- 51 -

surface showed that Al2O3 with small particle size is generally more efficient for the

enhancement of the impact strength, while the 100 nm particles proned to aggregation due to

their high surface energy deteriorating the impact strength. Composite filled with Al2O3 of

0.5 μm at content of 25 vol% showed the best synthetic properties. It is suggested that the

addition of nano-Al2O3 to HDPE would lead to good performance once suitably dispersed.

The thermal properties and combustion behaviour of new PE-hydrotalcites nanocomposites

were described by Costantino et al. 2005. Hydrotalcites were synthesized and then

intercalated with stearate anion with a long alkyl chain that played the role of compatibilizer

for the polyethylene chains.

The presence of inorganic filler shields PE from thermal oxidation, shifting the temperature

range of volatilisation towards that of thermal degradation in nitrogen, and brings to a

reduction of 55% in heat release rate during combustion.

In order to manufacture micro-porous, breathable films applicable to absorbent articles,

LDPE, LLDPE, and HDPE/zeolite composite films filled with zeolite, uncoated and coated

with stearic acid, were prepared (Kim et al. 2006). The mechanical, thermal, morphological,

and rheological measurements pointed out that the stearic acid showed a role of coupling

agent. Its incorporation on the zeolite surface improved the flexibility of polymer matrices.

In particular in LLDPE and HDPE based composites, the increase in the area of the air holes

and the decrease in the number of the air holes by merging among neighboring air holes was

prominent upon higher filler loading, therefore resulted in enhanced modulus, elongation and

impact properties.

Whereas, the declined values were observed in melting temperature (Tm), crystallization

temperature (Tc), and complex melt viscosity (Kim et al. 2006).

Studies on composite materials based on low-density polyethylene (LDPE) with different

weight percent of kaolin are present in literature (Hindryckx et al. 1996).

Shnean et al. 2008 analyzed some mechanical and physical properties such as tensile

strength, tensile strength at break, Young modulus, elongation at break, shore hardness and

water absorption at different weight fraction of filler (0, 2, 7, 10 and 15%). The addition of

filler increased the modulus of elasticity, elongation at break, shore hardness and impact

strength values; on other hand, the tensile strength and tensile strength at a break showed a

decreasing trend. Absorption test was carried out in water at different immersion times and

different composite .The results showed that after the addition of kaolin the absorption rate

exhibited a decrease. Calcinations kaolin filler produces better mechanical properties, than

grain kaolin fillers.

Ethylene polymerization was done with the aim to obtain a polyethylene based nano-

composite with nano-aluminum nitride using zirconocene as catalyst (Sohail et al.2012).

Results showed that the catalytic activity is maximum at a filler loading of 15 mg nano-

aluminum nitride. DSC and XRD results showed that the percentage crystallinity was also

marginally higher at this filler amount.

Morphology of the composite with 15 mg aluminium nitride is more fibrous as compared to

0 mg aluminium nitride and higher filler loading as shown by SEM images. In order to

understand combustibility behavior, tests were performed on micro-calorimeter. These


- 52 -

results showed a decrease in combustibility for the polyethylene nano-composites as the

filler loading increased.

Mechanical properties for Linear Low-Density Polyethylene (LLDPE)/org-clay based

nanocomposites prepared by melt processing were investigated by Durmus et al. 2008.

TEM study pointed out that the nano-composite samples showed a mixed morphology that

could be defined as some exfoliated layers, intercalated clay stacks, and two or three layered

tactoids present together within the samples.

Bentonite filled PP composites were prepared by melt-blending and the effect of

compatibilisers on mechanical, thermal, water absorption and morphological properties of

bentonite filled polypropylene based composites was investigated by Othman et al. 2006.

Two types of compatibilisers namely, Palm Oil Fatty Acid additive (POFA) and

Polypropylene Grafted-Maleic Anhydride (PPMAH) have been used with the aim to improve

the composites mechanical properties. The impact strength and elongation at break values

increased with the presence of both compatibilisers whilst, tensile strength and Young‘s

modulus improved only with the addition of PPMAH. Morphological investigation using

SEM revealed that the improvement in impact strength and elongation at break values was

due to the enhancement of the interfacial adhesion between bentonite and PP. The thermal

stability of bentonite filled PP improved with the incorporation of POFA and PPMAH. From

DSC thermograms, fusion enthalpy for both crystallisation (∆Hc) and melting (∆Hm)

processes of composites with compatibilisers have significantly reduced as compared to

control. The melting, cooling temperatures and crystallinity also reduced as compatibilisers

were added into bentonite filled polypropylene based composites.

Less percentage of water absorption has been observed in PP-bentonite system with PPMAH

(Othman et al. 2006).

Melt compounding was used to prepare conventional composites of montmorillonite clay and

PE as well as nano-composites of exfoliated montmorillonite platelets dispersed in a

maleated polyethylene (PE-g-MAn) matrix (Gopakumar et al. 2002). The extent of clay

platelet exfoliation in the PE-g-MAn nano-composites was confirmed by X-ray diffraction

and it resulted in a significant reduction of the degree of crystallinity and an increase of the

polymer crystallization rates. Studies of non-isothermal crystallization kinetics suggested

that the exfoliated clay promotes heterogeneous nucleation and two-dimensional crystallite

growth.

PE/clay composites behaved in a similar manner as conventional macrocomposites,

exhibiting modest increases in their rheological properties and Young‘s modulus.

Conversely, the nano-scale dimensions of the dispersed clay platelets in the nano-composites

led to significantly increased viscous and elastic properties and improved stiffness. This was

attributed to the high surface area between the polymer matrixes (Gopakumar et al. 2002

Introduction

- 53 -

1.6 Biodegradation of Fossil Fuel Based Polymers (ECOFLEX, PE and PS) in Soil-

Plant Systems (Avena sativa –Raphanus sativus)

The utilization of plastic mulch has played a major role in the increase in vegetable yield

production. The advantages of using mulch films are to control or increase the soil

temperature, maintain soil humidity, reduce germination time, provide early or out of season

crop production for better market value produce, reduce weeds and plant diseases, provide

efficiency in the usage of water and fertilizers, and most importantly, increase the quality of

horticultural products by avoiding the direct contact of the fruit with the soil (Lamont, 1999;

Espi et al., 2006).

Opaque mulching film prevents the passage of photosynthetically active radiation (PAR)

(Jayasekara et al. 1999), thus inhibiting weed growth, while transparent mulching films allow

the passage of the solar radiation, which increases soil temperature, thus improving the

cultivation cycle or providing soil sterilization following harvest (Immirzi et al. 2009).

Fig. 1.12. Pictures of the Black Biodegradable Films During and After the Growing

Season (Bar Length Equals 5 cm) (Kijchavengkul et al. 2008)

Figure 1.13. Pictures of the White Biodegradable Films During and After the

Growing Season (Bar Length Equals 5 cm) (Kijchavengkul et al. 2008).

http://it.wikipedia.org/wiki/Raphanus_sativus


- 54 -

Plastic mulching films, made mainly with low density polyethylene (LDPE), have

appropriate mechanical characteristics to assure their easy handling and fast installation,

functionality and resistance throughout the cropping cycle. Mulching films are designed with

suitable radiometric properties. The world consumption of LDPE mulching films in

horticulture is at present around 700000 tons per year (Espì et al. 2006).

Despite all these advantages, there are several major concerns about using mulch films which

are the costs of removal and disposal of the used plastics and environmental issues.

The use of plastic film in agriculture poses a major environmental problem due to high

molecular weight and hydrophobic properties of polyethylene. This gives to the film high

chemical stability, requiring about 100 years for its complete decomposition (Rutiaga et al.

2005).

In view of this, the introduction of biodegradable films (BPs) represents a really promising

alternative to the common (mostly polyethylene) plastic films for enhancing sustainable and

environmentally friendly agricultural activities (Gross and Kalra 2002). BPs can be degraded

by microorganisms in the natural environment.

The biodegradation test of all the mulch film samples in laboratory conditions was conducted

based on ASTM D 5338 standard (ASTM, 2003b) using a direct measurement respirometric

(DMR) system.

Biodegradable films can be degraded by microorganisms in the natural environment, thus

reducing the amount of waste ending up in landfills. To date, many aliphatic polyesters

which can be degraded in compost and moist soils, such as poly-(butylene succinate) (PBS)

and poly-(butylene succinate-co-butylene adipate) (PBSA) have been developed as

biodegradable films with excellent formability like polyethylene (Xu and Guo 2010).

The degradation of these materials, however, is gradual and cannot be easily controlled

(Kyrikou and Briassoulis 2007). Degradation speed of biodegradable mulch films in

agricultural fields is sometimes very slow, as it is largely affected by the environmental

conditions. The desirable practical biodegradable film is a programmed degradable plastic

that has adequate performance properties during its planned useful life time, and is 100%

post-use biodegradable.

If some efficient biodegradable film-degrading microorganisms were available, farmers can

use them or their enzyme to degrade the used biodegradable mulch films immediately after

use, thus enabling them to manage efficiently the schedule of their next planting. Also, they

will be able to use stronger plastic products that are not easily broken during use, and could

degrade them by enzyme treatment whenever necessary (Kitamoto et al. 2011).

Only few studies, however, have been published so far on the use of such microorganisms to

enhance the degradation of used biodegradable mulch films in soil environment (Kasuya et

al. 2009; Abe et al. 2010) isolated a fungal strain NKM1712 from soil, and observed weight

loss of poly butylene adipare-co-butylene terephtalate (PBAT) film with inoculation of this

strain in soil. Scanning electron microscopy (SEM) observation of degraded film on soil and

comparative studies of the biodegradable film-degrading ability of isolated microorganisms

demonstrated the contribution of fungi to microbial degradation of biodegradable films (Sang

et al. 2002; Tan et al. 2008; Kasuya et al. 2009). Recent investigation of microbial

communities in biodegradable-degrading compost by using direct DNA extraction from

http://www.sciencedirect.com/science/article/pii/S0045653507015287#bib3

Introduction

- 55 -

compost followed by cloning and sequencing showed that Ascomycota comprised the most

dominant group of microorganisms during the biodegradation of biodegradable films

(Sangwan and Wu 2008; Sangwan et al. 2009). Although biodegradable mulch films are

degraded in soil, it has been very difficult to isolate the effective biodegradable film-

degrading microorganisms from it. The farmer could be oriented to utilize a grass cover in

order to stimulate microbial biomass in soil and its enzyme activities; this could enhance

biodegradation of xenobiotics recalcitrant and biodegradable compounds at rhizosphere

zone. It is, in fact, well known that plants promote the degradation of organic compounds by

immobilization, removal, and promotion of microbial degradation.

Kuiper at al. (2004) and Newman and Reynolds (2004) published reviews on

phytodegradation of organic pollutants, at root level (rhizodegradation), and Dzantor (2007)

addressed the state of rhizosphere ―engineering‖ for rhizodegradation of xenobiotic

contaminants. Soil microflora plays, in fact, vitally important role during rhizoremediation of

xenobiotics (Johnsen et al. 2005; Semple et al. 2007).

Plants have many important functions in the stimulation of the microbial metabolism,

providing a carbon source for microorganism activity, transferring the oxygen from air to the

soil (Olson et al. 2003; Yu et al. 2006), and releasing root exudates which can serve as

substrates for the total microbial community activity, thus increasing the number of

microorganisms (Salt et al. 1998). Recent studies have confirmed that effectively plants are

able to stimulate soil microbial activity also in extreme environments (Doni et al., 2012;

Bianchi and Ceccanti, 2010; Benitez et al., 2010).

Together with the biotic factors, abiotic agents, such as solar radiant intensity, temperature,

humidity etc., are of great importance in controlling the environmental degradability of

biodegradable films.

1.6.1. Biodegradation of plastics in soil

Biodegradation is governed by different factors that include polymer characteristics, type of

organism, and nature of pretreatment. The polymer characteristics such as its mobility,

tactility, crystallinity, molecular weight, the type of functional groups and substituents

present in its structure, and plasticizers or additives added to the polymer all play an

important role in its degradation (Artham and Doble, 2008; Gu et al., 2000b).

During degradation the polymer is first converted to its monomers, and then these monomers

are mineralized. Most polymers are too large to pass through cellular membranes, so they

must first be depolymerized to smaller monomers before they can be absorbed and

biodegraded within microbial cells.

The initial breakdown of a polymer can result from a variety of physical and biological

forces (Swift, 1997). Physical forces, such as heating/cooling, freezing/thawing, or

wetting/drying, can cause mechanical damage such as the cracking of polymeric materials

(Kamal and Huang, 1992). The growth of many fungi can also cause small-scale swelling

and bursting, as the fungi penetrate the polymer solids (Griffin, 1980). Synthetic polymers,

such as poly(caprolactone) (Toncheva et al., 1996; Jun et al., 1994), are also depolymerized

by microbial enzymes, after which the monomers are absorbed into microbial cells and

biodegraded (Goldberg, 1995). Abiotic hydrolysis is the most important reaction for


- 56 -

initiating the environmental degradation of synthetic polymers (Göpferich, 1997) like

polycarboxylates (Winursito and Matsumura, 1996), poly(ethylene terephthalate) (Heidary

and Gordon, 1994), polylactic acids and their copolymers (Hiltunen et al., 1997; Nakayama

et al., 1996), poly (α-glutamic acids) (Fan et al., 1996), and polydimethylsiloxanes, or

silicones (Lehmann et al., 1995; Xu et al., 1998).

Generally, an increase in molecular weight results in a decline of polymer degradability by

microorganisms. In contrast, monomers, dimers, and oligomers of a polymer's repeating

units are much easily degraded and mineralized. High molecular weights result in a sharp

decrease in solubility making them unfavorable for microbial attack because bacteria require

the substrate to be assimilated through the cellular membrane and then further degraded by

cellular enzymes. At least two categories of enzymes are actively involved in biological

degradation of polymers: extracellular and intracellular depolymerases (Doi, 1990; Gu et al.,

2000b). During degradation, exoenzymes from microorganisms break down complex

polymers yielding smaller molecules of short chains, e.g., oligomers, dimers, and monomers,

that are smaller enough to pass the semi-permeable outer bacterial membranes, and then to

be utilized as carbon and energy sources. When the end products are CO2, H2O, or CH4, the

degradation is called mineralization (Frazer, 1994; Hamilton et al., 1995). It is important to

note that biodeterioration and degradation of polymer substrate can rarely reach 100% and

the reason is that a small portion of the polymer will be incorporated into microbial biomass,

humus and other natural products (Atlas and Bartha, 1997; Narayan, 1993). Dominant groups

of microorganisms and the degradative pathways associated with polymer degradation are

often determined by the environmental conditions. When O2 is available, aerobic

microorganisms are mostly responsible for destruction of complex materials, with microbial

biomass, CO2, and H2O as the final products. In contrast, under anoxic conditions, anaerobic

consortia of microorganisms are responsible for polymer deterioration. The primary products

will be microbial biomass, CO2, CH4 and H2O under methanogenic (anaerobic) conditions

(Barlaz et al., 1989).

1.6.2. Polyethylene

Synthetic polyolefins are inert materials whose backbones consist of only long carbon

chains. The characteristic structure makes polyolefins non-susceptible to degradation by

microorganisms.

However, a comprehensive study of polyolefin biodegradation has shown that some

microorganisms could utilize polyolefins with low molecular weight (Yamada-Onodera et

al., 2001). The biodegradation always follows photodegradation and chemical degradation.

Polyethylene is one of the synthetic polymers of high

hydrophobic level and high molecular weight. In natural form, it is not biodegradable. Thus,

their use in the production of disposal or packing materials causes dangerous environmental

problems (Kwpp and Jewell, 1992). To make PE biodegradable requires modifying its

crystalline level, molecular weight an mechanical properties that are responsible for PE

resistance towards degradation (Albertsson et al., 1994). This can be achieved by improving

PE hydrophilic level and/or reducing its polymer chain length by oxidation to be accessible

for microbial degradation (Bikiaris et al., 1999). The degradation of polyethylene can occur

by different molecular mechanisms; chemical, thermal, photo and biodegradation.

Some studies (Lee et al., 1991; Glass and Swift, 1989; Imam et al., 1992; Gu, 2003) have

assessed the biodegradability of some of these new films by measuring changes in physical

properties or by observation of microbial growth after exposure to biological or enzymatic

Introduction

- 57 -

environments, but mostly by CO2 evolution. Since polyethylene (PE) is widely used as

packaging material, considerable work not only on biodegradable polyethylene but also on

biodegradation of polyethylene has been conducted (Bonhomme et al., 2003; Wang et al.,

2004). The results of these studies indicated that polyethylene was biodegraded following

photodegradation and/or chemical degradation. Biodegradation of polyethylene is known to

occur by two mechanisms: hydro-biodegradation and oxo-biodegradation (Bonhomme et al.,

2003). These two mechanisms agree with the modifications due to the two additives, starch

and prooxidant, used in the synthesis of biodegradable polyethylene. Starch blend

polyethylene has a continuous starch phase that makes the material hydrophilic and

therefore, catalyzed by amylase enzymes. Microorganisms can easily access, attack and

remove this part. Thus the hydrophilic polyethylene with matrix continues to be hydro-

biodegraded. In case of pro-oxidant additive, biodegradation occur following

photodegradation and chemical degradation. If the pro-oxidant is a metal combination, after

transition, metal catalyzed thermal peroxidation, biodegradation of low molecular weight

oxidation products occurs sequentially (Bonhomme et al., 2003; EI-Shafei et al., 1998;

Yamada-Onodera et al., 2001). EI-Shafei et al. (1998) investigated the ability of fungi and

Streptomyces strains to attack degradable polyethylene consisting of disposed polyethylene

bags containing 6% starch. He has isolated 8 different strains of Streptomyces and two fungi

Mucor rouxii NRRL 1835 and Aspergillus flavus. In a study, Low density polyethylene

pieces buried in soil mixed with sewage sludge were examined microscopically after 10

months, fungal attachment was found on the surface of the plastic, indicating possible

utilization of plastic as a source of nutrient (Shah, 2007) The isolated fungal strains were

identified as Fusarium sp. AF4, Aspergillus terreus AF5 and Penicillum sp. AF6. The ability

of the fungal strains to form a biofilm on polyethylene was attributed to the gradual decrease

in hydrophobicity of its surface (Gilan et al., 2004).

The structural changes in the form of pits and erosions observed through scanning

electronmicroscopy indicated surface damage of PE incubated with Fusarium sp. AF4. That

suggested that the fungal strains, especially Fusarium sp. AF4, were able to adhere to the

surface of LDPE and can cause surface damage (Shah, 2007). In a study by Bonhomme et al.

(2003), SEM evidence confirmed that microorganisms (fungi) build up on the surface of the

polymer (polyethylene) and after removal of the microorganisms, the surface became

physically pitted and eroded. The surface of the polymer after biological attack was

physically weak and readily disintegrates under mild pressure. Otake et al. (1995) reported

the changes like whitening of the degraded area and small holes on the surface of PE film

after soil burial for 32 years. Biodegradation of LDPE film was also reported as 0.2% weight

loss in 10 years (Albertsson, 1980). Ohtaki et al. (1998) tested LDPE bottles exposed in

aerobic soil for over 30 years, and observed some evidences of biodegradation using SEM of

the degraded parts and as reduction in molecular weight by Time of Flight Mass

Spectrometry (TOF-MS). Yamada-Onodera et al. (2001) isolated a strain of fungus

Penicillium simplicissimum YK to biodegrade polyethylene without additives. UV light or

oxidizing agents, such as UV sensitizer, were used at the beginning of the process to activate

an inert material, polyethylene. Polyethylene was also treated with nitric acid at 80 °C for 6

days before cultivation with inserted functional groups that were susceptible to


- 58 -

microorganisms. Bonhomme et al. (2003) has reported that with the fungal activity,

polyethylene with a starting molecular weight in the range of 4000 to 28,000 was degraded

to units with a lower molecular weight of 500 after three months of liquid cultivation, which

indicated the biodegradation of that polyethylene. In a similar study the Polyethylene pieces

were treated by exposing it to UV light and also nitric acid. That pretreated polymer was then

applied to microbial treatment using Fusarium sp. AF4 in a mineral salt medium containing

treated plastic as a sole source of carbon and energy. An increase in the growth of fungus and

some structural changes as observed by FTIR, were observed in case of treated PE which

according to Jacinto indicated the breakdown of polymer chain and presence of oxidation

products of PE. Non-degraded polyethylene exhibits almost zero absorbancy at those wave

numbers (http://www.dasma. dlsu.edu.ph/offices/ufro/sinag/Jacinto.htm). Absorbance at

1710–1715 cm−1 (corresponding to carbonyl compound), 1640 cm−1 and 830–880 cm−1

(corresponding to −C=C–), which appeared after UV and nitric acid treatment, decreased

during cultivation with microbial consortia (Hasan et al., 2007). Typical degradation of PE

and formation of bands at 1620–1640 cm−1 and 840–880 cm−1 was also reported by

Yamada-Onodera et al. (2001), attributed to oxidation of polyethylene.

Overall, polyethylene degradation is a combined photo- and bio-degradation process. First,

either by abiotic oxidation (UV light exposure) or heat treatment, essential abiotic precursors

are obtained. Secondly, selected thermophilic microorganisms degrade the low molar mass

oxidation products to complete the biodegradation (Bonhomme et al., 2003).

1.6.3. Polystyrene

Polystyrene (PS) is a synthetic plastic used in the production of disposable cups, packaging

materials, in laboratory ware, in certain electronic uses. PS is used for its lightweight,

stiffness and excellent thermal insulation. When it is degraded by thermal or chemical means

it releases products like; styrene, benzene, toluene and acrolein. There are very few reports

on the biodegradation of polystyrene but the microbial decomposition of its monomer;

styrene, have been reported by few researchers (Tsuchii et al., 1977).

1.6.4. Aliphatic–aromatic (AAC) copolyesters

Biodegradable polyesters are one family of polymers that have been considered as

replacements for conventional plastic resins. Aliphatic polyesters such as the naturally-

occurring polyhydroxyalkanoate family of materials and the synthetic polycaprolactone have

been investigated as possible alternatives to traditional plastics, but their material properties

seriously affect the versatility of these materials (Reddy et al., 2003; Khanna and Srivastava,

2005; Oda et al., 1995). A more promising alternative is an aliphatic–aromatic copolyester of

1,4-butanediol, adipic acid and terephthalic acid, which has been commercialized under the

trade names Ecoflex_ and Eastar Bio_. The co-polyester is produced from the random

polymerization of the diester oligomers of adipic acid/butanediol, and terephthalic

acid/butanediol.

The degradation of this co-polyester has been investigated in compost environments or with

compost isolates Kleeberg et al., 1998; Witt et al., 2001). The experiments with compost

isolates were all performed at elevated temperatures with Thermobifida fusca (known

previously as Thermomonospora fusca) and a hydrolase isolated from this bacterial strain.

Results from these studies demonstrated rapid degradation of the co-polyester, with

essentially complete degradation having been achieved in 21 days in the presence of a

Introduction

- 59 -

readily available carbon source. However, numerous questions remain unanswered regarding

the degradation of the synthetic co-polyester under more moderate environmental conditions

and with microorganisms other than thermophiles. This is an important consideration

because it is likely that much of the co-polyester will be disposed of and degraded under

these mesophilic conditions rather than in compost at elevated temperatures.

In a more recent paper (Trinh Tan et al., 2008), a screening procedure was developed to

assess the biodegradability of the co-polyester at mesophilic environmental conditions and to

investigate the mechanism of biodegradation. Results showed that the aliphatic–aromatic co-

polyester could be degraded by a number of different microorganisms. However, after 21

days exposure to even the most promising cultures of pure microorganisms, only partial

degradation of the Ecoflex was accomplished and only a few samples showed visible signs

of degradation as loosely defined by the mechanical weakening of the films. Weight loss was

not as obvious as the visual degradation and suggested broader types of microbial attack. The

bacteria studied preferentially degraded the bonds between aliphatic components of the

copolymer and the rate of biodegradation of oligomers was appreciably faster than that for

the polymer chains. Using GC–MS techniques, degradation intermediates were identified to

be the monomers of the co-polyester. Gel permeation chromatography results suggested exo-

enzyme type degradation, where the microbes hydrolysed the ester bonds at the termini of

the polymeric chains preferentially.

1.7 Polyvinylics Biodegradation

The present global situation in the production of synthetic polymers accounts for almost 85-

90% represented by full carbon-backbone macromolecular systems (polyvinylics and

polyvinylidenics) (Chart 2004), and for about 35-45% of usage as one-way use items

production (disposables & packaging). Therefore it is reasonable to envisage a dramatic

environmental impact as attributable to the accumulation of plastic litter and waste

constituted by full carbon backbone polymers, which basically are recalcitrant to physical-

chemicals and biological degradation processes.

In opposition to ―hydro-biodegradation‖ process of natural and synthetic polymers

containing hetero atoms in the main chain (polysaccharides, proteins, polyesters, polyamides,

polyethers, polyurethanes), the mechanism of biodegradation of full carbon backbone

polymers requires an initial oxidation step as mediated or not by enzymes followed by the

fragmentation again mediated or not by enzymes with substantial reduction in molecular

weight. The functional fragments become then vulnerable to microorganisms present in

different environments with production under aerobic conditions of carbon dioxide, water

and cell biomass. In Figure 1.7 are outlined the general features of Environmentally

Degradable Polymeric Materials and Plastics that are classified into the specific sectors of

the Hydro-biodegradables and Oxo-biodegradables. Typical examples of the so called ―oxo-

biodegradable‖ polymers are represented by polyethylene, poly(vinyl alcohol), natural rubber

(poly 1,4 cis isoprene) and lignin (a natural heteropolymer) (Scott and Wiles 2001). The

major biodegradation mechanism of PVA in aqueous media, is represented by the oxidative

random cleavage of the polymer chains,. the initial step being associated to the specific

oxidation of methylene carbon bearing the hydroxyl group, as mediated by oxidase and

dehydrogenase type enzymes, to give -hydroxyketone as well as 1,3-diketone moieties. The

latter groups are susceptible of carbon-carbon bond cleavage promoted by specific -

diketone hydrolase, leading to the formation of carboxyl and methyl ketone end groups

(Sakai K, et al. 1986; Suzuki 1979).


- 60 -

1.7.1 The Role of Plastics in Agriculture

The fundamental role acquired by plastics in agriculture since the middle of the last century

(Albertsson, et al. 1994) was suggesting the definition of ―plasticulture‖ as a new practice

where the use of plastics for crops production and preservation can be considered as a routine

practice capable of increasing unitary yields, inducing earlier harvests, limiting the use and

release of herbicides and pesticides, as well as to improve the more efficient water resources

utilization[www.plasticulture.com], thus contributing significantly to the economic viability

of farmers worldwide (Lamont 1991). Accordingly conventional plastic films, are therefore

extensively used as coverings of greenhouses, low tunnels and mulching, where low density

(LDPE) and linear low density poly(ethylene) (LLDPE) films represents the most employed

polymeric materials (Briassoulis 2005; Espí, et al. 2006) accounting for an estimated

worldwide market of 2.0- 2.5 million tons per year in the agricultural segments.

The large utilization of these materials in certain agricultural regions is posing increasing

concerns about their collection and disposal (Decree 2006) after their service life. In

addition, visual pollution and detrimental effects on soil management and fertility have been

also claimed as problems connected to plasticulture These problems can be solved,

specifically for the agricultural plastic wastes, by using in situ biodegradable materials

having unitary costs comparable to those of conventional plastics. In this case, however, the

requirement for a controlled life time ranging between several weeks up to one year or more

depending upon the practical utilizations (i.e. mulching films or tunnels covering) is needed.

In keeping, since the second half of the 20th century, strategies to achieve low cost

environmental degradable polyolefins were undertaken (Guillet, et al. 1974; Voigt 1966).

The knowledge (G. Scott and Wiles 2001; N. C. Billingham and Calvert 1983) on thermal

and photolytic peroxidation mechanisms of poly(ethylene) and poly(propylene) constituted

the basis for the development of reengineered polyolefins susceptible to be oxidized and

fragmented when exposed to heat or light irradiation, with the aim to overcome the intrinsic

recalcitrance of polyolefins to biodegradation. It was ascertained that at the beginning of the

sequence of reactions leading to polyolefins peroxidation and their subsequent instability in

the environment is the generation of sensitising impurities during the thermal processing of

polyolefin resins (Scott 1993).

Carbonyl groups and hydroperoxides groups particularly were recognized as the most

effective sensitizing impurities capable of promoting the radical chain reactions leading to

the carbon chains cleavage in polyolefins (Scott 1993; Guillet and Norrish 1954; Hartley and

Guillet 1968; Carlsson et. al 1979; Scott 1981; Malaika et. al 1968; Heskins and Guillet

1968). In particular it was ascertained that the starting point is the formation of peroxyl

radical as a consequence of homolytic bond cleavage from shear stresses during extrusion in

the presence of oxygen, which is converted to hydroperoxide group by hydrogen abstraction

from vicinal methylene groups. The high reactivity of hydroperoxide groups under both heat

and UV exposure further promote an autoaccelerating cycle of interlocking reactions

involving formation/decomposition of radical groups which lead to the carbon chain scission

(molar mass reduction) and formation of several different oxidized groups. In the overall

peroxidation process of PE, the hydroperoxide groups decomposition is therefore considered

as the rate-determining step (Erlandsson 1997). Hence, molecules and additives which are

capable of enhancing hydroperoxide formation and decomposition to other radicals are active

pro-oxidants or pro-degradants since they accelerate the polyolefin chains oxidation and

successive cleavage.

Taking into account these evidences, major strategies to enhance the environmental

degradation and biodegradation of polyolefins have been focused on copolymerisation,

blending or grafting with functional polymers and compounds, as well as in the addition of

pro-oxidant additives. UV-absorbing carbonyl groups capable to accelerate the photo-

oxidation process can be introduced by copolymerizing ethylene and carbon monoxide or

Introduction

- 61 -

vinyl ketones (Harlan and Kmiec 1995; Guillet 1973), the latter strategy being the technical

process used in the production of Ecolyte® polyethylenes.

Another strategy to improve the environmental degradability of polyethylene and

polypropylene films is represented by the addition of pro-oxidants additives during their

conversion to plastic items. It has been in fact suggested that this alternative may provide a

more efficient control of the degradation rate, thus making the shelf life and useful life of the

polyolefin‘s compatible with a wide range of applications (Wiles and Scott 2006).

Most of the used pro-oxidant additives are represented by organic complexes of metals

capable of yielding two metal ions differing in the oxidation number by one unit

(Jakubowicz 2003; Weiland et al 1995). Several polymer soluble metal carboxylates and

acetylacetonates of Co3+ and Fe3+ are very effective photo-prooxidants capable to initiate

the degradation process through the metal salts photolysis to give under UV irradiation the

reduced form of the metal ion and a free radical.

The anion radical promote a fast hydrogen abstraction from the polymer and the relevant

formation of hydroperoxide. Afterward the general radical oxidation mechanisms of the

polyeolefins is thought to proceed being enhanced by the usual redox reactions between

hydroperoxides and metal ions FeX2, + ROOH > RO* + FeX2.OH (3).

Another class of pro-oxidant additives is represented by compounds capable to induce the

peroxidation of polyolefins by absorbing energy as heat. Also this class of additives is based

on the activity exerted by transition metal ions typically added to the final product in the

form of stearate or acetylacetonate organic ligands. The most employed cations are Mn2+

(Jakubowicz 2003) and Co2+ (Weiland et al 1995). Instead of Fe3+

complexes which play

the main role in photo-oxidation processes, Mn2+

and Co2+

are needed to accelerate the

radical chain reactions of polyolefin oxidation through the decomposition of hydroperoxides

and peroxides as induced by heat absorption (Koutny 2006) (Scheme 2).

An other type of ―photosensitizer‖ pro-oxidants is constituted by Fe(III) dithiocarbamates

and dithiophospates. These compounds initially acts as antioxidants by decomposing

hydroperoxides by an ionic mechanism (Malaika 1983; Malaika and Scott 1983), after that

the ligands are destroyed, thus releasing free transition metal ions which start to behave as

pro-oxidant. Hence, in these compounds both antioxidant and pro-oxidant properties are

coexisting. This characteristic feature has been exploited in order to finely control the

lifetime before the photo-oxidation commences according to the so called Scott-Gilead

technology which leads to the production and commercialization under the trade name of

Plastor of photodegradable polyethylene mulching films.

Several studies have therefore reported the significant reduction of molecular weight after

thermal and photo degradation of PE samples containing pro-oxidants (Albertsson et. al

1995; Albertsson et. al 1998), as well as the extraction, isolation and identification of

oxidized products, including carboxylic acids, ketones, esters and low molecular weight

hydrocarbons (Albertsson 1993; Khabbaz and Albertsson 2000). Carbonyl groups along the

carbon backbone are produced by additive-catalyzed hydroperoxide decomposition. A side

reaction of hydroperoxides is also the conversion in alkoxy macroradicals that in a similar

way of peroxy radicals may be transformed (hydrogen abstraction) to produce a carbonyl

group and a chain-end radical through chain scission. Accordingly it has been repeatedly

ascertained a straightforward relationship between the amount of chain scissions and the

number of carbonyl groups. For that reason, quantitative FT-IR analysis can be currently and

effectively used to predict the extent of abiotic photo- and thermal degradation of polyolefins

(Chiellini 2006). As a consequence of the radical oxidation processes and relevant chain

scissions, a fairly high number of degradation products containing functional groups have

been recognized during several investigations

Aliphatic carboxylic and dicarboxylic acids have been found to be the most abundant

degradation products which are formed during both photo and thermal oxidation of

additivated polyethylenes.


- 62 -

These intermediates can be also formed from the oxidation of other functional groups such as

primary alcohol and aldehydes deriving from hydroperoxide decomposition followed by

hydrogen abstraction or -scission, respectively, particularly during thermal aging. Also the

photolytical cleavage of keto groups through Norrish I mechanism (Scheme 1.3) may lead to

the formation of aldehyde that can be then oxidized to carboxylic groups. In keeping,

dicarboxylic acids have been found as the most predominant products formed during photo-

oxidation processes of PE especially after many days of irradiation (Albertsson 1995).

Since the ultimate environmental fate of ―degradable‖ polyethylenes has to be considered as

the results of the combining action of abiotic factors and microorganisms which suggested

the definition of ―oxo-biodegradable‖ materials in keeping with the processes of

biodegradation of lignin and natural rubber (Wiles and Scott 2006) . The evaluation of the

extent and rate of peroxidation of these materials represents indeed a powerful tool to predict

their potential biodegradation. Several studies have been therefore carried out with the aim of

investigating the mechanisms of photo and thermal oxidation of polyolefins containing pro-

oxidant additives. Nevertheless, most of these studies have been carried out under strictly

controlled laboratory conditions, as well as under extreme accelerating stressing conditions

that can not be considered as representative of the natural environments. In particular, only a

few investigations have been carried out by assessing the synergic effects of temperature,

humidity and/or sunlight exposure that usually are jointly operative in outdoor exposure

conditions.

In any case, by considering the general mechanism of radical oxidation of PE (Scheme 5),

the following parameters can be currently monitored during the abiotic degradation step

sketched for a PE blown film : 1) weight variation; 2) carbonyl index (COi); 3) wettability;

4) molecular weight variation; 5) fractionation by solvent extraction; In particular,

gravimetric analysis can be effectively used to appreciate the weight variation as a

consequence of the oxygen uptake during the early stage of oxidation, as well as the weight

loss due to the volatilization of low molecular weight fragments at prolonged stage of

thermal and photo degradation.

Another powerful tool for the quantitative and qualitative evaluation of the oxidation

processes is constituted by the carbonyl index (COi) as determined by FT-IR spectroscopy. It

has been repeatedly reported that the most of the degradation intermediates of PE

peroxidation are bearing carbonyl groups, therefore their concentration, as determined by

COi can be used to monitor the progress of degradation (Gugumus 1996).

In general these determinations are carried out on test films by recording the ratio of the

optical density of the carbonyl absorption band at 1640 - 1840 cm-1 range and the optical

density of the absorption band at 1463 cm-1 (CH2 in plane occurring in the vibrations -

scissoring ). In addition, FT-IR analyses provide also information on the presence during the

time of absorption attributable to of different carbonyl containing oxidized products such as

carboxylic acids (1712 cm-1), ketones (1723 cm-1), aldehydes (1730 cm-1) and lactones

(1780 cm-1) ( Khabbaz and Albertsson 2000).

The determination of wettability of film surfaces during the degradation tests by contact

angle measurements may provide useful information about the increasing polarity of the film

surfaces as a consequence of oxidation and formation of functional groups. These

information are also useful in order to predict the feasibility of microbial attack of PE films,

by taking into account that one of the reason that have been suggested to explain the intrinsic

recalcitrance of PE to biodegradation is the hydrophobic character hindering the adhesion of

microbial cells.

In terms of potential ultimate biodegradation (e.g. conversion to CO2 and H2O,

mineralization), the assessment of molecular weight variation is a fundamental task. It is in

fact suggested that from a theoretical point of view, PE in force of its structure as a straight

chain hydrocarbon, should be metabolized according to the biochemical pathway of linear

alkanes. On the other hand, it has been established that there is a dimensional limit (i.e

Introduction

- 63 -

molecular weight) for the n-alkanes utilization as carbon source by microorganisms. In this

connection, Haines and Alexander established that linear hydrocarbons with more than 44

(tetratetracontane) carbon atoms can not be metabolized by soil microorganisms (Haines and

Alexander 1974).

Recently, in a study carried out with single bacterial strains, this dimensional limit has been

moved to 0.72 kDa corresponding to 60 carbon atoms (Heat et. al 1997). In any case, these

limits are though to be related to the bacterial metabolism of n-alkanes that need the

accessibility of the methyl chain ends by extracellular oxidizing enzymes to start the

biodegradation process. Therefore, the rate and eventually the ultimate extent of

biodegradation of solid n-alkanes is strongly affected by the availability of -CH3 chain ends

susceptible of enzymatic oxidation. It is obvious that the chain ends present at the surface of

a solid alkane decrease with the increase of the molecular weight up to extremely low

numbers in the case of high molecular weight polyethylenes.

Finally, other information about the relationship between the level of oxidation as reached

during the abiotic stage of degradation of ―degradable‖ PE, the molecular weight reduction

as well as the potential to be biodegraded in the environment can be effectively achieved by

fractionation of pre-aged specimens submitted to solvent extraction. This practice may also

provide, especially if carried out by using solvents with different polarity, further

information on the relative amount of different classes (e.g. carboxylates, alkanes etc.) of

degradation products deriving from peroxidation and cleavage of PE chains.

Further to the evaluation of the abiotic oxidation degradable PE, the final step to be

investigated in order to achieve information on the ultimate environmental fate of the

partially oxidized materials is the assessment of biodegradation under different conditions.

The requirement of two steps, abiotic and biotic, in the degradation mechanism of oxo-

biodegradable plastic items has recently inspired the definition and approval of the ASTM

D6954-04 ―Standard guide for exposing and testing plastics that degrade in the environment

by a combination of oxidation and biodegradation‖.

This standard provides a framework to assess and compare the degree of degradation

attainable under controlled thermal and photo-oxidation tests with the degree of

biodegradation and ecological impacts in defined disposable environments after abiotic

degradation. In accordance the ASTM D6954-04 guide is divided in three tiers as sketched in

Figure 6.

i) accelerate aging in standard tests for both thermal and photo-oxidation processes

and determination of the degree of abiotic degradation (Tier 1),

ii) measuring biodegradation (Tier 2),

iii) assessing the ecological impact after this processes (Tier 3).

In order to exploit the Tier 1, the standard norm suggests to use test conditions for thermal or

photo-oxidation likely to occur in application and disposal environment for which the test

material is committed. In other words, accelerated oxidation should be carried out at

temperatures and humidity ranges typical of test material application and disposal

environment. Test materials resulting from Tier are therefore exposed to appropriate disposal

or use environments (soil, landfill, compost) in standard respirometric (biometric) test

methods in order to assess the rate and degree of biodegradation (Tier 2). Finally, any

residues of the analyzed materials, as deriving from both the abiotic oxidation stage and from

biodegradation tests must be submitted to ecotoxicity tests to demonstrate their ultimate

environmental compatibility (Tier 3).

1.7.2 Biodegradation of pro-oxidant-free polyethylene by natural occurring

microorganisms

The contemplation of the inherent capabilities of microorganisms to promote the

biodegradation and eventually the utilization of polyethylene (PE) as carbon source seems to


- 64 -

be more controversial nowadays that in fairly recent past years. Conflicting results have been

indeed obtained that are suggesting from one side the ability of specific microbial strains to

degrade high molecular weight PE, whilst many studies are yet claiming the inertness to

biological attack of this polyolefin and structurally alike polymers also when exposed to very

stressing and active microbial environments such as the composting windrows. Degradation

of PE by microorganisms has been generally monitored in terms of microbial growth,

biometry (e.g. biochemical oxygen demand or carbon dioxide emission, including 14CO2

generation from radio-labelled samples), sample weight loss, mechanical properties,

variation of structural features (e.g. molecular weight, spectroscopic features, mechanical

properties).

Preliminary investigations are dating back to the early 1960s. At that time, the increase of

microbial cell numbers was taken as an indication of PE assimilation by bacteria in

comparison with low molar mass paraffins (Jen-hou and Schwartz 1961). From these studies

it was suggested that several bacteria can utilize as carbon source low molecular mass PE

fractions slightly below 5000, whereas no microbial activity was detected on higher Mw

fractions. Potts and co-workers assessed that linear paraffin with Mw below 500 were

utilized by different microorganisms (Potts 1972). Similar results were obtained later by

Albertsson and Banhidi that recorded the utilization of short oligomeric fraction of HDPE by

microorganisms after 2 years biodegradation experiments (Albertsson and Banhidi 1980).

Indeed, it can be suggested from a theoretical point of view that since PE is a nominally

straight-chain hydrocarbon, it should be metabolized according to the biochemical pathway

holding true for linear alkanes (Fig.1.14). On the other hand, it has been established that

there is a molecular weight upper limit for the utilization of n-alkanes as a carbon source by

microorganisms. Haines and Alexander established that linear hydrocarbons with more than

44 carbon atoms (tetratetracontane) cannot be metabolized by soil micro-organisms ( Haines

and Alexander 1974). More recently in a study carried out by using single bacterial strains

(Heat et al. 1997), this dimensional limit has been extended to 720 Dalton corresponding to

an hydrocarbon chain constituted by 60 carbon atoms. In any case these limits are thought to

be related to the bacterial metabolism of n-alkanes that need the accessibility to methyl chain

ends by extra cellular oxidizing enzymes to start the biodegradation process.

RCH3 RCH2OH RCHO RCOOH

H3CRCH3 H3CRCOOH HOCH2RCOOH OCHRCOOH HOOCRCOOH

RCH2CH2CH3 RCH2CH(OH)CH3 RCH2COCH3 RCH2OC(O)CH3 RCH2OH + CH3COOH

RCH3 RCH2OOH RCHO

RCH2OH

RCOOH

RCOOCR

Terminal oxidation (Pseudomonas)

Diterminal oxidation (Candida)

Subterminal oxidation (Nocardia)

Finnerty's pathway (Acinetobacter)

Figure 1. 14. Microbial oxidation pathways of n-alkanes.

Introduction

- 65 -

The first step is known as hydroxylation ( -oxidation) which give rise to the corresponding

primary alcohols which are further enzymatically oxidized to aldehyde and hence to

carboxylic acids. The resulting carboxylated n-alkanes can be metabolized according to the

β-oxidation process in analogy with the catabolism of fatty acids. Thus, the rate and

eventually the ultimate extent of biodegradation of solid n-alkanes can be strongly affected

by the availability of CH3 chain ends susceptible to enzymatic oxidation. It follows that the

number of chain ends present at the surface of a solid n-alkane decreases with the molecular

weight increase and hence with extremely low values in the case of high molecular weight

polyethylene.

On the other hand, it can not be at least hypothetically ruled out that oxidizing biological

processes other than the -oxidation of terminal methyl groups such as the random chain

cleavage as mediated by dehydrogenation/oxidation leading to carbonyl groups might play

an important role in the biodegradation of PE.

Taking into account these suggestions Kawai and her collaborators tried to establish a

numerical simulation model for the biodegradability of PE starting from experimental data

relevant to the biodegradation of PE-wax having 2900 Mw and 1100 Mn, respectively

(Kawai et al. 2002; Kawai et al. 2004). The computational model for the numerical

simulation was set up regarding to two main factors:

i) sample weight loss due to -oxidation;

ii) fast consumptions of low molecular weight fractions. Both factors were

substantiated by experimental data.

In particular the increase of both Mw and Mn of PE wax was observed after cultivation as an

indication of the microbial consumption, in the meanwhile the time profile of molecular

weight variation suggested the fast assimilation of the lower Mw fractions, as well as the

gradual decrease of assimilation rates with increase in molecular weight. By applying the

numerical model, which was validated by experimental results, to the biodegradation of PE,

the authors provide suggestions supporting the terminal oxidation and -oxidation as the

main processes involved in the microbial degradation of PE. In addition, by using this

approach it was possible to distinguish between the process of biodegradation as mediated by

bacteria such as Sphingomonads or Aspergillus sp. and Penicillium sp. fungi. It was

therefore evidenced that bacteria exhibited higher biodegradation rates that were attributed to

the higher affinity toward PE of Gram-negative bacteria cell walls with respect to the more

hydrophilic chitin walls of fungi (Kawai et al. 2004). Sphingomonas species were thought to

metabolize PE wax, preferentially with Mw below 2000, throughout primary terminal

oxidation followed by -oxidation with enzymatic systems located in the cell membrane

fraction, thus suggesting for the transport of oxidized PE wax into the periplasmic space

through outer membranes (Kawai 1999).

Regarding the capabilities of single microbial species to attack PE as carbon substrate, only a

few reports are so far available. Nevertheless, in the recent years evidences in the occurrence

of soil microorganisms directly involved in the biodegradation processes of PE have been

reported by Ohtake and collaborators (Ohtake et al. 1998; Watanabe et al. 2009).

1.7.3 Preliminary data or studies

Free radical oxidation, as induced by thermal and / or photolytic pre-abiotic treatment

constitutes the first step for promoting the fragmentation (Figure 1.15) and eventual

biodegradation of both LDPE and LDPE-containing pro-oxidant/pro-degradant additives.

This can be accomplished by monitoring the initial variation of sample weight, molecular

weight and other structural parameters (tensile strength, degree of crystallinity, level of

oxidation by FTIR and wettability by contact angle). Biodegradation is then observed when

degraded oxidized polymer fragments are exposed to biotic environments.


- 66 -

Figure 1.15. Fragmentation in outdoor exposure of PE Bags containing pro-oxidant/pro-

degradant additives

Additionally, the required degree of macromolecular breakdown for the microbial

assimilation of polyethylene to occur is substantiated by the observation of the propensity to

biodegradation of lower molecular weight hydrocarbon molecules. It has been demonstrated

that linear hydrocarbon having molecular weight below 500 (Potts 1972) or n-alkanes up to

tetratetracontane (C44H90, Mw = 618) (Haines and Alexander 1974) can be utilized as

carbon source by microorganisms. The degradation of higher molecular weight untreated

high-density poly(ethylene) with molecular weights up to 28,000 by a Penicillium

simplicissimum isolate it has been also reported (Yamada et. al 2001) even though the extent

of fungal attack of poly(ethylene matrix) has been monitored by physicochemical tools (FT-

IR, HT-GPC), and by growth-proliferation assays in agar plates containing the indicated PE

sample. No respirometric data have been unfortunately reported.

As the carbonyl group is representative of most of the oxidation products (carboxylic acids

ketones, aldehydes and lactones) (Hakkarainen and Albertsson 2004) of the oxidative

degradation of polyethylene, the concentration of carbonyl groups, as determined by the

carbonyl index (COi) can be used to monitor the progress of oxygen uptake and degradation

(Chiellini et al. 2006) . COi determinations have been therefore utilized in order to compare

the thermal oxidation behavior of test samples under dry and 75% relative humidity

conditions at 55 and 70°C (Chiellini et al. 2006).

First of all, the oxidation of the poly (ethylene) matrix in the thermally aged samples has

been clearly ascertained by the FT-IR spectroscopy. The increasing absorption and

broadening within time of the band in the carbonyl region has been recorded in all the

samples aged at 70°C . Overlapping bands corresponding to acids (1712 cm-1

), ketones (1723

cm-1

), aldehydes (1730 cm-1

) and lactones (1780 cm-1

) have been also observed, thus

indicating the presence of different oxidized species. Among these carboxylic acids and ester

groups have been shown to be produced in the early and later stages of polymer matrix

oxidation, respectively (Albertsson et al 1995). The increase of specimen weight up to a 4-

5% of the original value, as well as the increase of wettability of the thermally treated films,

Introduction

- 67 -

50,0

60,0

70,0

80,0

90,0

100,0

Conta

ct a

ngle

(°)

day 0 day 4 day 7 day 10 day 15

as determined by contact angle measurements, further demonstrated the polymer oxidation

(Figures 1. 16, 1. 17).

Figure 1. 16. Weight Variation Profile of LDPE Sample of LDPE Samples at Increasing

Level of Oxidation.

Figure 1. 17. Contact angle and density change Containing Pro-Oxidant Upon Aging at

70ºC

Thermally treated samples have been also characterized by HT-GPC, the recorded molecular

weight and molecular weight distribution (ID) have been compared with the COi values

detectable at the same time of thermal ageing (Chiellini et al. 2006). A drastic decrease of

Mw below 5 kDa and significant reduction of the ID have been thus recorded after a few

days of thermal degradation under both dry and 75% RH conditions. In HT-GPC

chromatograms relevant to LDPE samples retrieved at longest thermal degradation exposure,

thus having the highest level of oxidation, elution peaks having a bimodal shape and very

low (1.7 kDa) Mw fractions have been also detected. (Table 1.2)


- 68 -

Table 1.2 Molecular weight analysis of thermally oxidized LDPE-containing pro-

oxidant/pro-degradant additives

Figure 1.18. Relationship between the level of oxidation expressed as COi and Mw in

thermally oxidized LDPE-containing pro oxidant/pro-degradant additives.

The amount of oxidized degradation intermediates extractable by acetone, which can be

considered more prone to be diffused in the environment, has been shown to positively

correlate to the level of oxidation In addition, the relationship between the Mw and COi was

found to fit a mono-exponential trend (Figure 1.18).

Accordingly, COi values may be used in order to predict the rate of Mw decrease as a

function of the oxidation extent. Moreover, the recorded trend is in agreement with a

statistical chain cleavage mechanism, repeatedly suggested in the photophysical and thermal

degradation of polyolefin (Scott 1994; Gugumus 2001) thus reaching fairly high quantity

corresponding to 25-30% specimen weight (Figure 19).

Time COi Mw ID

(days) (kD)

0 0.61 39.4 4.24

1 1.14 19.5 2.96

2 2.32 9.7 2.59

9 5.44 4.5 1.27

Introduction

- 69 -

Figure 1. 19. Acetone extractable fraction of thermally oxidized LDPE-containing pro-

oxidant/pro-degradant additives

The recorded data have to be considered as partially representative of the overall amount of

low Mw fractions produced during the thermal oxidation of the test material. Indeed

molecular weights as low as 0.8-1.6 kDa for the acetone extracts of thermally treated

samples have been recorded by SEC. It is also worth noting that at higher level of oxidation,

increasing amounts of the extractable fractions are recorded, and in the meantime these

fractions are characterized by a very low Mw (Figure 14) (Chiellini et al. 2006). Therefore,

the reported data suggest that cross-linking reactions does not affect notably the oxo-

degradation behavior of the analyzed samples, which seems to proceed with cleavage of the

macromolecules up to low molecular weight fractions capable to be assimilated by

microorganisms (Volke-Sepŭlveda et al. 2002; Potts 1972; Kawai et al. 1999). Indeed, earlier

studies have demonstrated the microbial utilization as carbon source of the oxidation

products formed during the thermal and photo-oxidation of polyethylene (Arnaud et al.

1994). Accordingly, in a respirometric experiment, aimed at evaluating the biodegradation

behavior of low molar mass aliphatic hydrocarbons in soil, including linear docosane, (C22),

branched 2,6,10,15,19,23-hexamethyltetracosane, (Squalane - C30), and docosandioic acid,

(C22), a fairly high rate and extent of mineralization (70%) of the acetone extractable

1.49 kD

1.08 kD

1.06 kD

0.89 kD

0

5

10

15

20

25

30

COi 0.63 COi 2.24 COi 4.82 COi 5.44

Extract (%) Mw Extract (kD)


- 70 -

fraction from a thermally oxidized LDPE-TDPA sample was recorded (Chiellini et al. 2003).

(Figure 1.20)

Figure 1.20. Biodegradation profiles in soil burial respirometric tests of acetone extract of

pre-oxidized LDPE-TDPA samples and low molar mass aliphatic hydrocarbons.

This suggests that, similarly to low molar mass hydrocarbons, the oxidized fragments from

LDPE can be rapidly biodegraded in natural compartments (soil and surface waters).

There are several studies dealing on the evaluation of biodegradation of LDPE samples

containing pro-oxidant and natural fillers that show only limited and slow conversion to

carbon dioxide. The mineralization rate from long-term biodegradation experiments of both

UV-irradiated samples, non-pretreated, and additive-free LDPE samples, in natural soils

indicates more than 100 years for the ultimate mineralization of polyethylene (Ohtake et al.

1998).

In a soil burial test, carried out in the presence of forest soil as incubation media, the

biodegradation profile of a thermally pretreated LDPE-TDPA sample was monitored during

830 days of incubation (Chiellini et al. 2003). The recorded mineralization kinetic was

characterized by the presence of a first exponential step that approached a 4 % degree of

biodegradation within 30 days of incubation. Afterward, a prolonged (120 days) stasis in the

microbial respiration was recorded. At approximately five months of incubation it was noted

that a further and marked exponential increase in the biodegradation profile took place, thus

reaching the highest extent of biodegradation 63.0 %) after 85 weeks of incubation (Figure

1.21). This two-step mineralization profile of thermally fragmented LDPE-TDPA samples

has been repeatedly observed either in soil and mature compost biodegradation tests. In

contrast with previous studies (Ohtake et al. 1998; Albertsson et al. 1988 ), very large degree

of mineralization (70-80 %) have been then recorded, even though in a relatively long time

frame (more than 800 days) (Chiellini et al. 2003).

Figure 1.21. Biodegradation in soil burial respirometric tests of pre-oxidized LDPE-TDPA

samples

A substantial increase in the carbon-carbon unsaturation, as revealed by the increase of the

double bond index from 0.55 at the beginning to 0.88 after 62 weeks of incubation, was

recorded.

This can be attributed to enzymatically driven dehydrogenation (Ohtake et al. 1998;

Albertsson et al. 1998). Hence it is likely that the macromolecular cleavage of thermal-

Introduction

- 71 -

oxidized LDPE-TDPA can be achieved concomitantly by abiotic oxidation and biotic

enzymatically promoted action. Furthermore a dramatic change in the fingerprint region of

the FTIR spectrum between 1300 and 950 cm-1

was observed with increasing the incubation

time probably attributable to the presence of lower molecular weight fragments.

Therefore, it seems that the ongoing abiotic and biotic degradation of thermally degraded

LDPE-TDPA polymer bulk is occurring during the incubation in forest soil with the

production of large amount of degradation intermediates capable to be assimilated by the soil

microflora. This might explain the presence of the prolonged dormant phase (stasis), as well

the second, more pronounced, exponential phase repeatedly observed in the biodegradation

kinetic profiles of these materials recorded in soil burial respirometric tests.

A similar approach has been utilized in order to evaluate the propensity to biodegradation of

photo-degradable LLDPE samples for mulching purposes (Corti et al. 2010).

As expected, control sample without pro-oxidant/pro-degradant additives also showed a

steady increase in the COi but at a significantly slower rate compared to the level of

oxidation achieved in films with additives. The observed increase of COi in LLDPE films

under sunlight is attributed to the initiation of chain scission as a result of photo-oxidation

producing fragments in the form of shorter, more readily crystallizable molecules (Chiellini

et al. 2006; Chiellini et al. 2003; Sepúlveda et al. 1999).

The mere exposure to sunlight led to a considerably lowered value in the onset degradation

temperatures (Ton) in films with additives when compared to unexposed controls during

TGA analysis. As repeatedly ascertained, sunlight-induced aging resulted in the formation of

low molecular weight products

Photodegradable LLDPE films (LLDPE-TD1 and LLDPE-TD2) pre-exposed to sunlight for

93 days were submitted to biodegradation in the presence of single fungal strains previously

isolated from thermally oxidized LDPE fragments confined in forest soil.

A vibrant growth of mycelia on the surface of the film aged in sunlight was apparent

indicating a robust fungal metabolic activity. The growth of mycelia on the film surface

showing cracks in the film developing around the edge of mycelium were also visible at a

higher magnification (Figure 1.22) (Corti et al. 2010).

a) b) Figure 1. 22. SEM micrographs of the fungal mycelium mycelium growing on the surface of

outdoor exposed LLDPE-TD2 specimens (a). The b is a higher magnification of A, showing

fungal hyphae colonizing the film surface

A similar behavior has been also observed by Weiland et al. (Weiland et al. 1995) for

thermally oxidized PE films that were further treated in compost for a period of time. Film

surfaces were increasingly colonized by fungi as examined by SEM, and the increase in

fungal presence on the surface was accompanied by a decrease in molecular weight of the

film, providing a strong evidence of fungal bioassimilation of the amorphous regions of


- 72 -

thermally oxidized films.

The FT-IR spectra of the test and control samples reveal the prominence of absorption bands

attributed to the C=C within the polymer chain in films incubated with fungal strains.

Additionally, new absorption bands in the finger print region between 1700 – 1100 cm-1

of

the spectra and a considerably elevated -OH absorption peak in the OH stretching vibration

region, were also noticeable in the samples incubated with fungi for 65 days but not in the

controls. The appearance of new bands can be then associated with the formation of

metabolites generated by the fungal attack on the pre-oxidized fragments.

Mineralization tests were conducted to learn if soil fungi could utilize the breakdown

products derived from the oxidized LLDPE film as carbon source. To do this, pre-oxidized

LLDPE-TD1 film was incubated on a solid-agar media with soil fungi in the presence of

glucose as secondary carbon source. The control consisted of pristine LLDPE films that had

not been pre-exposed to sunlight.

The pre-oxidized LLDPE films submitted to the fungal exposure experienced an

enhancement in the amount of breakdown products as reflected by the increase in the FT-IR

peak intensities typical of several oxidized carbon functional groups. Interestingly, these

increases were only visible in pre-oxidized films incubated with fungi but not in controls.

This fact once again is pointing towards the synergistic impact of biotic and abiotic factors in

achieving efficient LLDPE degradation.

The information pertaining the level of photo-oxidation required to achieve an effective and

sustained biodegradation in LLDPE, is crucial for the design of LLDPE-based products and

prediction their environmental fate. Nowadays information with respect to synergistic effects

of microbial/enzymatic attack and physical-chemical parameters in promoting the

degradation of partially oxidized LLDPE can be provided. The data suggest that the

degradation of oxo-biodegradable LLDPE samples is enhanced by the synergistic action of

both abiotic and biotic factors after their initial oxidation by exposure to direct sunlight.

The data substantiate the validity of oxo-biodegradable polymers and confirm that PE films

with pro-oxidant/pro-degradant additives are environmentally degradable by means of a

combination of abiotic and biotic factors. Thus, several conclusions can be drawn from the

fungal biodegradation studies on LLDPE films with pro-oxidant/pro-degradant additives:

(a) Prolonged sunlight exposure causes considerable oxidation in these films leading to their

deterioration and to the production of degradation products,

(b) Soil borne fungi are fully capable of utilizing degradation products as a carbon source,

(c) Presence of an additional carbon source (under co-metabolic conditions) could stimulate

fungal metabolic activities to further promote the degradation of the LLDPE polymer matrix,

possibly mediated by hydrolytic enzymes produced by fungi and

(d) More effective degradation of films with pro-oxidant additives could be achieved by a

combined action of abiotic and biotic factors.

Objective

- 73 -

2. OBJECTIVES

The research activity of my Doctorate Thesis was aimed at the assessment of biodegradation

extent in different solid media (soil and compost) and river water of blends and composites

based on poly(lactic acid) (PLA) and a poly(hydroxyalkanoate) copolymer consisting of

97.5% 3-hydroxybutyrate (HB) and 2.5% of 3-hydroxyvalearte (HV) indicated from now on

as PHBV.

The biodegradability tests have been carried out on a few selected blends and composites of

PHBV used as continuous phase in comparison with paper as positive control and the PHBV

matrix alone. The PHBV polymer was blended with new additives such as chain extenders,

organic and/or inorganic fillers and the produced composites and blends were investigated

for their biodegradation propensity upon a thorough characterization of the physical and

mechanical properties of the samples.

The study highlighted the effect of each component on the kinetic profiles of the biobased

polymeric formulations.

The PHBV sample behaves as a rigid and brittle material due to its high crystallinity (Scott

2002, Maiti et al. 2007). In order to improve its mechanical properties, blends and

composites were prepared and submitted to a thorough physical chemical characterization.

Three different strategies were adopted:

1) Plasticization of PHBV with compatible and biodegradable plasticizer together with

nucleating agents

2) Blending of PHBV with biodegradable polymers poly (butylen adipate terephthalate)

(Ecoflex)

3) Combination of components applied in the 1 and 2 strategic approaches.

A reactive additive (Joncryl ADR-4368C) was used for its ability in controlling the polymer

molecular weight eventually occurring during its processing in the melt. It promotes indeed

the reactions between the epoxide groups of the additive and the end capping groups present

in the PHBV original chains. Until now, the utilization of PHAs in the production of

commodity plastic items is extremely limited, due to their high production cost with respect

to conventional petrochemical polymers. In particular the preparation of blends and

composites of PHAs with conventional polymers and agro-industrial wastes respectively

constitutes a strategic approach aimed at overcoming the drawback bound to the relatively

high cost/performance of the PHAs. For that reason a powder of hazelnut shells was used as

organic filler either alone or with other additives. Such filler increases by a few degrees the

melting temperature of the final composite.

Specimens of the prepared blends and composites were submitted to biodegradation tests in

solid media (compost and soil). The biodegradation of PHBV organic composites in soil

seems be favored by the presence of hazelnut shell powder, but Joncryl appears to delay

somewhat the propensity to biodegradation under the same environmental conditions. In

compost medium similar trends to those assessed in soil medium, were detected.

The propensity of synthetic and biobased plastics products, such as Ecoflex, Ecovio, PHBV

and PLA and their composites, to biodegradation in soil medium, was followed also from a

biological point of view. Different polymers as powder samples (polyethylene, polystyrene,

Ecoflex and cellulose) at concentration of 1% by weight, were added to the soil, with the aim


- 74 -

to monitor the meso-scale experiment consisting of soil sample carried out after one and 45

days from the experiment set up. Chemical, biochemical and biological parameters were

used to evaluate the soil conditioning during the polymer biodegradation. Furthermore, two

terrestrial plants, oat (Avena sativa) and red radish (Raphanus sativum), were used to

evaluate the phytotoxicity of the polymers. For this purpose, after 35 days from the

experiment set up, 100 seeds of each plant species were sown uniformly in each pot. The

upper growth was checked at the end of the experiment. Polymers stimulated soil metabolic

activity, thus indicating their possible biodegradability in soil. These effects were more

evident in Radish treatments than in Avena, suggesting the higher sensitivity of radish to the

polymers treatment.

Meanwhile, the depolymerase activity of polymers was also determined as further

investigation to know the enzyme activity effect on the biobased polymers PHB and PLA.

Experiment

- 75 -

3. EXPERIMENTAL PART

3.1. Materials

3.1.1. Reagents and Solvents

The reagents and solvents employed in the experimental part and their respective

suppliers are listed below:

Potassium hydroxide pellets, technical grade (KOH), was supplied by CARLO

ERBA (Rodano-Milan-Italy).

Hydrochloric acid (HCl) volumetric standard, 0.1N, analytical grade, was supplied

by J.T.Baker (Italy).

Barium chloride (BaCl2), technical grade was purchased from CARLO ERBA

(Rodano-Milan-Italy).

3.1.2. Joncryl ADR-4368-CS

Joncryl ADR-4368-CS is a chain extender suitable for food packaging applications, supplied

by BASF, The Chemical Company (Germany) as a white granulate or a powder

characterized by a granulometry similar to the sugar one (Figure 3.1).

Figure 3.1. Powder of Joncryl ADR-4368CS.

Its chemical structure (Figure 3.2) shows the presence of epoxy groups able to react with the

terminal carboxylic and hydroxy groups of the polymer chains preventing the formation of

degradation products and oligomers with short chain during the reactive extrusion process.

Figure 3.2. Joncryl ADR-4368C Chemical Structure.


- 76 -

Table 3.1 reports the physical features of the material.

Table 3.1. Physical Features of Joncryl ADR-4368CS.

Property Value

Density at 25°C (g/cm3) 1.08

Mw (Da) 6800

Tg (°C) 54

Equivalent Epoxy Weight (g/mol) 285

Ton (°C) 320

Figure 3.3 shows the IR spectrum for the chain extender Joncryl ADR-4368CS.

Figure 3.3. Spettro IR per il Joncryl ADR-4368CS.

3.1.3. Hazelnut Shells

The hazelnut shells (Figure 3.4), supplied by Ferrero S.p.A (Italy), were grinded in a mill

Brabender Wiley, with a tri-phase engine (power = 0.75 kW), equipped with four static, six

strong blades and four interchangeable sieves (0.5 mm, 2 mm, 3 mm, 4 mm).

The material was sieved until the obtainment of a particle dimension of 39 micron: this value

results to be suitable for a good dispersion of the organic filler inside the polymeric matrix.

(a) (b)

Figure 3.4. Raw Hazelnut Shells (a) and After Grinding (b).

Experiment

- 77 -

The typical chemical composition for hazelnut shells is collected in Table 3.2, whereas

Figure 3.5 shows the IR spectrum for the obtained ligno-cellulosic material.

Table 3.2. Hazelnut Shells Chemical Composition.

Components (%) Hazelnut Shells

Lignin 45.5

Hemicellulose 9.5

Cellulose 24.97

Hydro-soluble Substances 9.9

Ashes 1.27

Fats & WAxes 1.2

Figure 3.5. IR Spectrum for the Ligno-cellulosic Sieved Below 39 Micron.

3.1.3.1. Lignin Functionalization

The functionalized lignin is obtained by using an acid catalyzed Fisher esterification.

The reactions have been carried out according to the Dean-Stark method. Typically, the

reactions have been carried out in 500 ml two necked round bottom flask equipped with a

Dean-Stark trap and a 20 ml addition funnel, by suspending 5.0 g LN in 100 ml toluene.

After 1 h at reflux a solution of 2.5 g of stearic acid (STEA) and 100 mg of p-toluensulfonic

acid monohydrate (pTSA), both supplied by Sigma-Aldrich, in 20 ml toluene was added

drop-wise and the reaction mixture was maintained other 5-7 h at reflux. After cooling under

N2 the suspended solid was recovered by vacuum filtration, washed with 50 ml of acetone to

remove the un-reacted aliphatic acid and finally dried under vacuum up to constant weight

before characterization.

In this way, the lignin becomes more hydrophobic and compatible with the polymeric

material.

3.1.4. Organophilic Clay (Dellite 72T)

Dellite 72T, supplied by Laviosa Mineraria S.p.A (Italy) as white powder, is a nano-clay

deriving from a natural purified montmorillonite modified with quaternariy ammonium salt.

Its density is 1.7 g/cc while the humidity can be reaching a value of 3%. This clay is directly

mixed in the composite with the aim to improve the O2, CO2 and water vapour barrier

properties.

Figure 3.6 shows the IR spectrum for this material.


- 78 -

Figura 3.6. IR Spectrum for Dellite 72T.

3.1.5. Perkalite F100

Perkalite F100 is a sinthetic clay organically modified and consisting of a double hydoxide

layers (LDH), named hydrotalcite. The polymer loading with Perkalite makes favourable the

clay delamination at nano-scale level, improving the thermo-mechanical, the barrier and

rheological properties of the polymeric materials.

3.1.6. Poly(3 hydroxy-co-3 valerate) (PHBV)

PHBV chemical structure is reported in Figure 3.7 while in Table 3.4 are summarized the

main thermal and mechanical properties of PHBV and commercial PHA copolymers,

compared with polypropylene and low density polyethylene (LDPE).

Figure 3.7.. Chemical Structure for Poly(3hydroxybutyrate – co – 3-hydroxyvalerate)

(PHBV).

O CH CH2 C

O

O CH CH2

CH2CH3CH3

C

O

* *

nm

Experiment

- 79 -

Table 3.4. PHB and Copolymers, Polypropilene (PP) and Low Density Polyethylene (LDPE)

Physical Properties[Scott 2002].

Property PHB 20V1)

6HA2)

PP LDPE

Melting Temperature, (˚C) 175 145 133 176 110

Glass Transition Temperature (˚C) 4 -1 -8 -10 -30

Cristallinity, % 60 ng ng 50 50

Density (g/cm3) 1,25 ng ng 0,91 0,92

E module, MPa 3,5 0,8 0,2 1,5 0,2

Tesile Stress, MPa 40 20 17 38 10

Strain at Break, % 50 680 400 600 400

1) poly(3-hydroxybutyrate-co-20 mol% 3-hydroxyvalerate)

2) poli(3-hydroxybutyrate-co-6 mol% HAs) [HAs (hydroxyalkanoate) = 3 %

3-hydroxydecanoate, 3% 3-hydroxydodecanoate, < 1% 3-hydroxyoctanoate, < 1%

5-hydroxydodecanoate, ng= negligible].

Table 3.5. General Properties for PHBV ENMAT Y1000.

Property Value Determination Method

Molecular Weight (Mw,

kDa)

450 GPC in chloroform

Polydispersity Index (IP) 1.5 GPC in chloroform

Appearance White

Powder

Visual observation

Melting

Temperature(Tm, °C)

167 DSC Mettler Toledo DSC822°

Standard Method:

1st heating from 25°C until to 200°C, withan

heating rate of 10°C/min and 80 ml/min N2

Isotherm for 3 min. at 200°C and 80 ml/min N2

Cooling from 200°C until to -50 °C with a

cooling rate of 10°C/min and 80 ml/min N2

Isoterma 1 min. a - 50°C 80 ml/min N2

2nd heating dfrom -50°C until to 200°C, with an

heating rate of 10°C/min and 80 ml/min N2

Glass Transition

Temperature (Tg, °C)

3-4 DSC Mettler Toledo DSC822

Cristallinity (%) 75 Xc = (ΔHm/ΔHm100%)100 where ΔHm PHBV100% cristalline =

146 Jg-1

Valerate Content (mol %) 2 1H-NMR spectrum Varian Gemini VRX 200

Onset temperature

(Ton, °C)

258 TGA


- 80 -

Figure 3.8. Picture of Commercial PHBV in powder form.

Figures 3.9-3.11 show all the characterizations (IR,DSC, NMR) for PHBV ENMAT.

Figure 3.9. IR Spectrum of PHBV ENMAT Sample.

Figure 3.10. DSC Thermograms of PHBV ENMAT Sample recorded at Different Scanning

Conditions (2nd Heating).

Experiment

- 81 -

Figure 3.11. 1H-NMR Spectrum of PHBV ENMAT Sample.

3.1.7. Poly(lactic acid) (PLA)

PLA was supplied by Proplast (Alessandria-Italy) as opaque granules (Fig. 3.12b) and the

polymer chemical structure is reported in Figure 3.12a.

(a) (b)

Figura 3.12. PLA Chemical Structure (a) and powder Shape (b).

The D isomer content, as indicated by NatureWorks, is comprised in the range of 1.2-1.6%.

The molecular weight is below 160 kDa and the polidispersity index (Mw/Mn) is 2.31.

Figure 3.13 shows the IR spectrum for pure PLA sample.

O CH

CH3

C

O

OHH

n


- 82 -

Figure 3.13. IR Spectrum fof PLA Sample.

3.1.8. Poly(butylene adipate-co-terephthalate) (Ecoflex)

Ecoflex® FBX 7011 is a biodegradable, statistical, aliphatic-aromatic copolyester consisting

of 1,4–butanediol, adipic acid and terephthalic acid monomers (Figure 3.14), supplied by

BASF The Chemical Company (Germany) as an white powder.

Figure 3.14. Ecoflex Chemical Structure.

It has properties (Table 3.6), similar to LDPE, that make it suitable for film extrusion.

Table 3.6. General Features for Ecoflex Sample.

Property Value

Density (g/cm3) 1.25-1.27

Melt Flow Rate (g/10 min)

(MFR, 190°C and 2.16 Kg)

2.7-4.9

Melt Volume Rate (mL/10 min)

(MFR, 190°C and 2.16 Kg)

2.5-4.5

Melting Temperature (Tm, °C) 110-120

Shore Hardness (D) 32

Vicat Point (°C) 91

Trasparency (%) 82

E module (N/mm2) 95-80

Tensile Strength (N/mm2) 35-40

Ultimate Strength (N/mm2) 36-45

Elongation (%) 560-710

Failure Energy (J/mm) 24

Oxygen Transmission Rate (cm3/ m

2d*bar) 1400

Water Vapour Transmission Rate (g/m2*d) 170

Figure 3.15 reports the IR spectrum for Ecoflex sample.

HO C

O

CH24

C

O

O CH2 O4

C

O

C

O

OH

Experiment

- 83 -

Figure 3.15. IR Spectrum of Ecoflex Sample.

3.1.9. Ecovio

Ecovio F Blend C2224, supplied by BASF Chemical Company (Germany) as white granules,

is a biodegradable co-polyester consisting of Ecoflex and PLA (45% by weight).

This material results suitable for packaging applications by flexible and compostable films

production.

Table 3.7 shows the general features of Ecovio F Blend C2224.

Table 3.7. General Features of Ecovio F Blend C2224 Sample.

Property Value

Melting Temperature (Tm, °C) 140-155 (PLA)

110-120 (Ecoflex)

MVR (mL/10 min)

(190°C, 2.16 Kg)

< 2.5

MVR (mL/10 min)

(190°C, 5 Kg)

< 6.5

Vicat Point (°C) 68

E module (N/mm2) 520-750

Tensile Strength (N/mm2) 27-35

Ultimate Strength (N/mm2) 27-36

Elongation (%) 250-320

Oxygen Transmission Rate (cm3/ m

2d*bar) 600

Water Vapour Transmission Rate (g/m2*d) 92


- 84 -

Figure 3.16 shows the IR spectrum of Ecovio sample.

Figure 3.16. IR Spectrum of Ecovio Sample.

3.2. Preparation of the Sample

3.2.1. Pre-blend

The composites were prepared by blending all the components manually and these pre-

mixtures were then dried in an oven at 80°C overnight (in order to remove the major amount

of moisture). The blends and composites compositions are collected in Table 3.8. In

particular, the blends and composites loaded with the polymeric chain extender Joncryl (J)

were dispersed at room temperature in a mixer at 2 rpm for a time of 35 min.

3.2.2. Melt blending

The obtained PHBV and PLA samples (Tables 3.8-3-9) were melt-blending in an internal

mixer chamber (55 cc) connected to a Brabender plastograph (Germany) (Figure 3.17).

The processing conditions were 30 rpm, residence time 7 min to 170oC and mixture weight

of 30 g for PHBV based blends and composites whereas for the PLA ones, were: 60 rpm,

residence time 7min at 180°C, mixture weight 40 g.

Figure 3.17. Brabender-Plastograph Apparatus

Experiment

- 85 -

Table 3.8. Composition of Organic, Inorganic and Hybrid Composites Based on PHBV.

Sample code PHBV

(% wt)

Lignin

(% wt)

(LN)

Joncryl

(% wt)

(J)

Inorganic

(% wt)

(D) (LHD)

PHBV 100 - - -

PHBV + J0.2 99.8 - 0.2 -

PHBV + J2 98 - 2 -

PHBV + J5 95 - 5 -

PHBV + LN5 95 5 - -

PHBV + LN10 90 10 - -

PHBV + LN20 80 20 - -

PHBV + LN10 + J5 85 10 5 -

PHBV + LN20 + J5 75 20 5 -

PHBV + LDH3 97 - - 3

PHBV + D3 97 - - 3

PHBV + D6 94 - - 6

PHBV + D3 + J2 95 - 2 3

PHBV + LN10 + D3 87 10 - 3

PHBV + LN20 + D3 77 20 - 3

PHBV + LN10 + D3 +

J2

85 10 2 3

PHBV + LN20 D3 + J2 75 20 2 3

PHBV + LNF10a)

90 10 - -

Table 3.9. Composition of Organic and Hybrid Composites Based on PLA.

Sample Code PLA

(%-wt)

Lignin

(LN)

(%-wt)

Joncryl

(J)

(%-wt)

PLA 100 - -

PLAJ0.2 99.8 - 0.2

PLAJ5 95 - 5

PLALN10 90 10 -

PLALN20 80 20 -

PLALN10J5 85 10 5

PLALN20J5 75 20 5

3.2.3. Press

The mixtures coming from Brabender had been processed by using a press. About 5-8 g of

blend were compression moulded in a laboratory mould press Collin (model Collin P 200E)

with a water cooling system to obtain round films of width around 300-500 m and diameter

of 12 cm.

The plate of the press has dimensions about 196x196 mm and maximum compression force

about 125 kN. Moulding conditions were constituted by an heating cycle until to 170-180°C


- 86 -

for 5 minutes by using a pressure of 200 bar following by a cooling cycle until room

temperature for 8 minutes.

3.3 Characterization of the Samples

3.3.1. Thermogravimetric Analysis (TGA)

TGA analyses were performed with a Mettler Thermogravimetric Analyzer TA500 equipped

with a Mettler TG50 furnace and a Mettler M3 microbalance.. About 10 mg of the samples

were heated from 30°c to 700°C and heating rate of 10°C/min, in air atmosphere (flux about

80 ml/min) and nitrogen atmosphere (about 60 ml/min). The onset temperature (Ton

), at

which the material starts to decompose, was determined as the temperature corresponding to

the tangent‘s intersection on both sides of the decomposition trace. The residue was

evaluated as the residual weight at the end of the experiment.

3.3.2. Differential Scanning Calorimetry (DSC)

The prepared samples (5-10 mg) were analyzed by using a differential scanning calorimeter

Mettler-Toledo, DSC Model-822 equipped with a sensor FRS5 connected to a STARe

software. The samples were placed in 40 μl aluminium pans, while an empty one was used as

reference. The temperature calibrations were performed utilizing indio, iron and zinc as

standards.

The standards were used in order to perform enthalpy (∆H) calibration.

The DSC thermograms were recorded according to the following scheme:

Heating from 25 °C to 200 °C, 10 °C/min @ 80 ml/min (N2 flux)

Isotherm @ 200 °C for 3 min. @ 80 ml/min (N2 flux)

Cooling from 200 °C to -50 °C, 10 °C/min @ 80 ml/min (N2 flux)

Isotherm @ - 50°C for 1 min. @ 80 ml/min (N2 flux)

Heating from -50 °C to 200 °C, 10 °C/min @ 80 ml/min (N2 flux)

3.3.3. Transmision Fourier Transform Infrared Spectroscopy (FTIR)

Fourier transformed infrared (FT-IR) spectroscopic characterization of the analyzed

materials was carried out with a Jasco model 410 spectrophotometer. The spectra were

recorded as an average of 16 scans with a resolution of 2 cm-1

.

3.3.4. Nuclear Magnetic Resonance (1H-NMR)

1H-NMR spectra were recorded with a Varian Gemini VRX 200 from solution of the

polymer samples in CDCl3. This characterization allows us the assessment for the structural

features in terms of monomeric units‘ composition of the polymer samples.

3.3.5. Gel Permeation Chromatography (GPC)

The molecular weights, comprised in the linear range 200-2.000.000 Da, were determined by

using an HPLC cromatograph, mod. Jasco PU-1580, connected with two spectrofotometric

detectors (λ = 260 nm), Jasco 830-RI for the infrared frequences and Perkin-Elmer LC-75 for

the ultraviolet ones and equipped with two microcolumns (5 microliters) PLgel.

Chloroform stabilized with amylene were employed as eluent with a flux of 1.0 ml/min.

Experiment

- 87 -

The average molecular weight for the selected samples was calculated by considering the

calibration curve, which was obtained for standards monodisperse polystyrene samples.

3.3.6. Determination of Total Organic Carbon (TOC) and Total Nitrogen (TN)

Total organic carbon (TOC) and total nitrogen (TN) were determined by RC- 412

MULTIPHASE CARBON DETEMINATOR (RC-412 LECO®, ST. JOSEPH. MI 49085-

2396, U.S.A) and FP-528 PROTEIN/NITROGEN (Leco corporation), respectively.

To test the total organic carbon (TOC) of original polymeric samples, EDTA (EDTA. CAL

IBRATION SAMPLE; Part No. 502-092; CARBON %, 41.01±0.13; HYDROGEN %,

5.56±0.02; NITROGEN, 9.58±0.02; LECO CORPORATION. U.S.A ) was used as

calibration standard.

3.3.7. PH and Electrical Conductivity

The pH and electrical conductivity were determined on the aqueous extracts (1 hour at room

temperature under shaking) using the 672 TITROPROCESSOR (Thermo) and the ION

SELECTIVE ELECTRODES (Electron Corporation N-1017969), respectively.

3.4. Experimental Setup for Biodegradation Experiments

3.4.1. Soil Burial Biodegradation Tests

A soil burial respirometric test specifically set up from ISO 17556 (17556-03 ―Plastics –

Determination of the ultimate aerobic biodegradability in soil by measuring the oxygen

demand in a respirometer or the amount of evolved carbon dioxide‖) and ASTM D5988

(ASTM D5988 - 03 ―Standard Test Method for Determining Aerobic Biodegradation in Soil

of Plastic Materials or Residual Plastic Materials After Composting‖)standard test in order

to test polymeric materials with low or moderate propensity to biodegradation, was utilized

to investigate the ultimate biodegradation behaviour of PHBV based samples. In this test

procedure the soil is partially substituted with a hygroscopic inert mineral substrate (perlite)

that ensure satisfactory incubation conditions and in the mean time a more favourable ―signal

to noise ratio‖ resulting in improved accuracy particularly when limited carbon dioxide

emissions are expected from the mineralization processes of the test samples (Chiellini E.

and Corti A. 2003).

A natural forest soil sample (Table 3.10), sieved at 1 mm in order to remove any plant and

root debris, was used as incubation substrate because its richness in microbial population

capable to metabolize a wide spectrum of organic compounds including aromatic

hydrocarbons.


- 88 -

Table 3.10. Soil Sample Properties.

Property Value

Electrical conductivity (dS m-1

) 0.15

pH 6.71

Total Nitrogen (TN, %) 0.16

Total Organic Carbon (TOC, %) 1.46

Total Inorganic Carbon (TIC, %) 0.01

Holding Water Capacity (WHC, %) 20.0

Electrical Conductivity (distilled water: soil, 5:1); TN, Total Nitrogen; TOC, total Organic

Carbon; TIC, Total Inorganic Carbon; WHC, Water Holding Capacity.

The tests were carried out in cylindrical glass vessels (Biometer Flask) (750 ml capacity)

containing a multilayer substrate in which defined amounts of forest sandy soil (10 g) sample

were mixed with 15 g of perlite and supplemented with 10-15 ml of 0.1% (NH4)2HPO4

solution. Finally the mixtures were sandwiched between two layers consisting of 20 g perlite

and wetted with 30 ml distilled water.

Filter paper (FP) was used as reference compounds with the aim to validate the adopted test

procedure.

Test materials and reference compound were tested at approximately 50 mg/g dry soil ratio,

in duplicate runs. All the cultures were incubated at 25°C in the dark.

Figures 3.18 and 3.19 collect the pictures showing the starting material and the final

soil/perlite mixture, respectively.

Figure 3.18. Starting Materials and Final Pre-Treated Soil.

Figure 3.19. Pre-Treated Soil (Blend of Soil, Perlite and Water).

Experiment

- 89 -

Under the adopted conditions, the biodegradation behaviour was assessed for PHBV based

composites prepared with a ligno-cellulosic material derived from the grinding of Hazelnuts

shell (organic filler), Dellite 72T (organically modified montmorillonite as inorganic filler)

kindly supplied by Laviosa Mineraria S.p.A (Italy), and special additives like polymeric

chain extender, Joncryl ADR-4368C (Basf-Germany).

In Table 3.11 the PHBV based samples compositions and the relevant sample amount used

for the biodegradation test, are reported. The particle size for the PHBV composites was < 2

mm (mesh n° 10). Figures 3.20 and 3.21 are showing the biodegradation set up design and

the final sample shape of the PHBV composites, respectively.

Figure 3.20. Biodegradation Experimental Setup (Respirometric Apparatus).

Figure 3.21. Polymer Test PHBV Composite Samples.


- 90 -

Figure 3.22. Mineralization Test System for Biodegradation.

Experiment

- 91 -

Table 3.11. Sample Composition for the Biodegradation Test in Soil.

Test Sample Code Description (weight %) Shape Sample 1

(mg)

Sample 2

(mg)

PHBV PHBV powder 382.4 380.1

PHBV-LN20-D3-J2 PHBV+20%

Lignin+3%Dellite+2% Joncryl

powder 380.7 380.3

PHBV-LN20 PHBV+20% Lignin powder 381.5 380.4

PHBV-LN20-J50 PHBV+20% Lignin+50% Joncryl powder 380.8 380.0

PHBV-LN20-D3 PHBV+20% Lignin+3% Dellite powder 380.1 380.5

PHBV-D3-J2 PHBV+3% Dellite+2% Joncryl powder 380.8 381.1

PHBV-LN10-D3-J2 PHBV+10% Lignin+3%

Dellite+2% Joncryl

powder 380.3 382.6

PHBV-LN10 PHBV+10% Lignin powder 381.7 380.0

PHBV-LNF10 PHBV+10% Functionalized

Lignin

powder 384.1 381.6

PHBV-D3 PHBV+3% Dellite powder 380.8 384.7

PHBV-J5 PHBV+5% Joncryl powder 380.3 382.4

PHBV-LN10-J5 PHBV+10% Lignin+5% Joncryl powder 381.3 382.7

PHBV-LN10-D3 PHBV+10% Lignin+3% Dellite powder 381.2 384.2

Cellulose Cellulose powder powder 382.2 381.6

LN: Lignin from Hazelnuts shell; D: Dellite (inorganic component); J: Joncryl (Chain

extender useful for polyesters); LNF: Functionalized lignin with stearate groups.

3.4.2. Mature Compost Respirometric tests

Mature compost respirometric tests specifically set up from ISO 14855 standard (ISO 14855-

99 “Plastics – Evaluation of the ultimate aerobic biodegradability and disintegration of

plastics under controlled composting conditions – Method by analysis of released carbon

dioxide) in order to test polymeric materials with low or moderate propensity to

biodegradation, was utilized to investigate the ultimate biodegradation behaviour of PHBV

and PLA test materials (Chiellini E. and Corti A. 2003).

The tests were carried out in cylindrical glass vessels (Biometer Flask) (750 ml capacity)

containing a multilayer substrate in which defined amounts of mature compost from organic

solid waste (Table 3.12), sieved at 1.0 mm (7 g dry weight) sample were mixed with 15 g of

perlite. Finally the mixtures were sandwiched between two layers consisting of 20 g perlite

and wetted with 30 ml distilled water. Filter paper (FP) as reference compound was used in

order to validate the adopted test procedure.


- 92 -

Table 3.12 Compost Parameters.

Parameters U. M. Value Limit(*) Methods

Humidity at 105 ℃ % 29.5 <50% s.t.q. UNI 10780/98

pH unit pH 7.3 6-8.5 EPA 9045D 2002

Ratio C/N - 27.5 <50 UNI 10780/98

Organic Corban (as C) % s.s. 35.5 >20 UNI 10780/98

Humic acids + Fulvic

acids (as C)

% s.s. 7.3 >2.5 UNI 10780/98

Organic Natrogen (as N) % s.s. 87.4 >80% Nitrogen tot. UNI 10780/98

Total Nitrogen % s.s. 1.29 - UNI 10780/98

Salinity meg/100g 11.8 - UNI 10780/98

Cadmium mg/kg s.s. <0.1 1.5 UNI 10780/98

Mercury mg/kg s.s. <0.1 1.5 UNI 10780/98

Nickel mg/kg s.s. 18.7 100 UNI 10780/98

Sodium mg/kg s.s. 500 - UNI 10780/98

Lead mg/kg s.s. 7.9 140 UNI 10780/98

Copper mg/kg s.s. 24.2 230 UNI 10780/98

Zinc mg/kg s.s. 34.6 500 UNI 10780/98

Chromium Hexavalent mg/kg s.s. <0.01 0.5 UNI 10780/98

Plastic, Glass and Metals>

2mm

% s.s. <0.01 0.5 UNI 10780/98

Lithoid aggregates> 5 mm % s.s. 0.45 5.0 UNI 10780/98

Test materials and reference compound were tested at approximately 25 mg/g dry mature

compost ratio, in duplicate runs. All the cultures were incubated at 58 ± 2°C in the dark.

The pre-treated compost was stabilized until the evolution of carbon dioxide was comprised

between 50 and 150 mg/g of solid in 10 days according to the international norm ISO 14855-

99.

In a first step the biodegradation in the process of mature compost was carried out without

carbon dioxide monitoring for screening purposes by analyzing the physical changes of the

samples as well as the weight loss within the time of the most representative samples related

to PHBV and PLA based composites. The tests in compost were carried by placing the

sample on a layer of a pre-treated compost. The pretreated compost was prepared in a similar

way as the biodegradation test in soil. The control parameters in this case were the humidity

inside the chamber (must be 50%) and the ratio compost/Perlite 1:2 by weight. In Figure 3.23

the biodegradation setup design for qualitative experiment in mature compost is depicted.

The respirometric biodegradation test in mature compost was carried out by analyzing the

propensity to mineralization of PHBV and PLA based composites prepared with a ligno-

cellulosic material derived from the grinding of Hazelnut shells (organic filler) and special

additives like polymeric chain extender, Joncryl ADR-4368C.

In Table 3.13 the composition and shape for PHBV and PLA relevant based composites

submitted to the respirometric test, are collected. The particle size for the PHBV and PLA

Experiment

- 93 -

composites was < 2 mm (mesh n° 10). The biodegradation test set up for compost was

similar to the biodegradation set up in soil (see Figure 3.23).

Figure 3.23. Biodegradation Setup for Compost tests (for Screening Purpose).

Table 3.13. Composition and Shape for Selected PHBV and PLA Based Composites.

N Test Sample Code Description

(% in wt.)

Shape Sample 1

(mg)

Sample 2

(mg)

1 PHBV PHBV film 146.4 134.1

2 PHBV-J5 PHBV + 5 % Joncryl film 145.0 149.2

3 PHBV-J0.2 PHBV + 0.2 % Joncryl film 148.9 167.3

4 PHBV-LN20 PHBV + 20 % Lignin film 148.3 142.5

5 PHB-LN20-J5 PHBV +20 % Lignin +5 %

Joncryl

film 146.1 148.3

6 PLA PLA film 165.0 154.0

7 PLA-J0.2 PLA + 0.2 % Joncryl film 156.0 158.0

8 PLA-J5 PLA + 5 % Joncryl film 148.0 145.0

9 PLA-LN20 PLA + 20 % Lignin film 156.0 147.0

10 PLA-LN20-J5 PLA +20 % Lignin +5 %

Joncryl

film 157.0 142.0

3.4.3. River Water respirometric Test

The biodegradation test was carried out in accordance with the OECD 301 Guideline for

testing of chemicals and in particular with the OECD 301 B method ―CO2 evolution

(Modified Sturm Test)‖ The test was carried out in 300 ml Erlenmeyer flasks equipped with

silicone rubber stoppers hanging 40 ml capacity plastic vials filled with CO2 absorbing KOH

solutions (Figure 3.24). Each flask contained 100 ml of salt solution as incubation medium

having the following composition per liter of distilled water: KH2PO4 85 mg, K2HPO4 218

mg, Na2HPO4 334 mg, (NH4)2SO4 10 mg, NH4NO3 10 mg, CaCl2 36 mg, MgSO4·7H2O 23

mg, and FeCl·6H2O 0.3 mg, pH 7.4±0.2 A river water sample was used as microbial

inoculum at 10% by volume ratio.


- 94 -

All the test materials were supplied to the microbial culture as sole carbon sources at 0.01-

0.02% by weight concentration.

Test flasks were then incubated at room temperature (25°C) in the dark on a rotatory shaker

(120 rpm). All the runs were carried out in duplicate. Every 5 -7 days CO2 absorbers,

containing 20 ml of KOH solution (0.05 N) will be retrieved and replaced with fresh KOH

solution in known aliquots. The retrieved KOH solutions were back-titrated with 0.1 N HCl

and the amount of adsorbed CO2 was computed.

At the end of the test all the test materials were withdrawn from aqueous cultures, carefully

washed and characterized by gravimetric analysis (e.g. weight variation), FT-IR

spectroscopy and thermal analysis (TGA and DSC).

Figure 3.24. Schematic Representation of Aqueous Medium Biodegradation Tests.

3.4.4. Carbon Dioxide Determination and Evaluation of the Degree of Biodegradation in

Respirometric Tests

The evaluation of biodegradation degree in the respirometric test is based on CO2

determination in the headspace of the culture vessels (biometer flask). CO2 evolution, inside

a sealed biometer, is a consequence of the microbial assimilation of the test sample, and it

can be trapped in each test vessel by means of 40 ml of 0.05N KOH solution contained in a

beaker set in the test vessel. The absorbing solution was back-titrated with 0.1N HCl at time

intervals. The addition of 1 ml of barium dichloride (BaCl2) 0.5N before the titration in one-

tenth proportion respect to the overall volume of the absorbing alkaline medium, was

necessary for the carbonate anions precipitation as an insoluble barium carbonate (BaCO3)

salt. Phenolphthalein was used as indicator. All the tests were carried out in duplicate.

Subsequently, biometer was recharged with a fresh KOH solution and immediately closed.

Eventually distilled water (20 to 25 ml) was added into the flask in order to control the

humidity inside the vessel. This procedure cycle was repeated periodically up to the end of

experiment. The above procedure is based on the following reactions:

Carbon dioxide uptake by KOH solution

CO2 + 2 KOH→K2CO3 (soluble) + H2O

Insoluble barium carbonate formation

K2CO3 + BaCl2→BaCO3 (insoluble) + 2 KCl

Experiment

- 95 -

To determine complete or ultimate aerobic biodegradability (mineralization to CO2), it is

necessary to know the initial amount of organic carbon (TOC) in the sample and the relevant

theoretical amount of CO2 (ThCO2).

The amount of TOC was calculated as follow:

CS = TOCS * WtS TOC=Cs*Ws

Where CS is the % amount of carbon in the sample, TOCS is the relative amount of total

organic carbon in the sample at time 0 and WtS the weight of sample.

The extension of biodegradation defined as mineralization percent was calculated by

subtracting the accumulated CO2 content evolved into the biometers of sample (CO2)S from

that of blank (CO2)B. The difference found is divided by the theoretical amount of CO2

(ThCO2) as calculated on the basis of the carbon content of the test sample

Biodegradation (%) = 100*[(CO2) S - (CO2) B]/ThCO2

All the biodegradation rate calculation are based on the European standard EN 14046-03:

Packaging - Evaluation of the ultimate aerobic biodegradability of packaging materials

under controlled composting conditions - Method by analysis of released carbon dioxide.

3.5. Specific Biotests to Assess Eco-toxicity of Biodegradable Polymer Materials

Ecoflex, PE, PS in Soil-Plant systems

In the present investigation, the extent of biodegradation and potential eco-toxicological

effects of synthetic biodegradable biomaterials were investigated under laboratory conditions.

3.5.1 Materials

3.5.1.1. Soil and Plants used in the experiments


- 96 -

The characteristics of the soil used for the laboratory experiment are reported in Table 3.14.

Table 3.14. Chemical, Biochemical and Microbiological Parameters of the Soil Used in the

Experiment

Soil characteristics

Original soil umidity (%) 3.26±0.13

pH 6.93±0.27

Electrical conductivity (dS m-1

) 114±1.4

Total organic carbon (%) 2.50±0.03

Total Nitrogen (%) 0.323±0.01

NO3- (mg kg

-1) 234±6

NH4+ (mg kg

-1) 3.43±0.72

Available Cu (mg kg-1

) 77.8

Available Zn (mg kg-1

) 17.48

Available K (mg kg-1

) 323

Available Mg (mg kg-1

) 108

AvailableFe (mg kg-1

) 19.4

Available P (mg kg-1

) 27.5±3.8

Total Cu (mg kg-1

) 64±7

Total Zn (mg kg-1

) 58±23

Total Ni (mg kg-1

) 63.6

Total Pb (mg kg-1

) 26.7

Total Cr (mg kg-1

) 41.9

Total Cd (mg kg-1

) 0.3

Total K (mg kg-1

) 6076±1067

Total Mg (mg kg-1

) 6890±583

Total Fe (mg kg-1

) 8395±1097

Total cultivable microbial population (UFC g-1

) 5.34E+07±7.31E+06

Pseudomonas (UFC g-1

) 4.1E+05±4.9E+02

β-glucosidase activity (mgPNP kg-1

h-1

) 580

Dehydrogenase activity (mgINTF kg-1

h-1

) 2.75

The selected plants were:

1. Avena sativa (oat);

2. Raphanus sativus (red radish).

3.5.1.2. Testing materials

The following polymers were selected:

1. Polyethylene (PE): Polimeri Europa Riblene FL30, CIBA specialty chemical oxo-

biodegradable LLDPE, thermally degraded

2. Polystyrene (PS): Polimeri Europa powder, EPI-INC. oxo-biodegradable PS,

thermally degraded

3. Ecoflex: BASF – Ecoflex ® FBX 7011 powder (Figure 3.14)

4.

Figure 3.14. Ecoflex Chemical Structure. HO C

O

CH24

C

O

O CH2 O4

C

O

C

O

OH

Experiment

- 97 -

4. Cellulose

5. Poly-(3hydroxy)butyrate

3.5.2. Experimental Setup

3.5.2.1. Degradation in a soil-plant system

The experiment at mesoscale level was carried out using pots with a diameter of 25 cm and a

height of 10 cm (mesocosms) containing 1 kg of soil.

The different polymers as powder at the final concentration of 1% were added to the soil.

The pots were incubated under controlled temperature, light and humidity. The moisture

content of each soil–biomaterial mixture was adjusted to 50% of water holding capacity. The

monitoring of the mesoscale experiment consisted of soil sampling carried out after 35 (T0)

and 45 days (T1) from the experiment set up.

Each soil sample was a composite of three sub-samples mixed, homogenized, sieved (2 mm)

and stored dried at room temperature until chemical analysis and stored at 4°C until

biological analysis.

In addition, two terrestrial plants, oat (Avena sativa) and red radish (Raphanus sativus), were

used to evaluate the phytotoxicity of the biomaterials. For this purpose, after 35 days from

the experiment set up, soil was sampled (T0) and characterized, then 100 seeds of each plant

species were sown uniformly in one of the two replicate pots and all mesocosms were

maintained under controlled temperature, light and humidity for further ten days. The plant

growth was checked at the end of experimentation (45 days-T1); at this time the plants were

eradicated, washed and the dry weight of shoots and roots and the plant length were

determined.

3.5.2.2. Biodegradation of PHB in soil

The experiment at microscale level was carried out using pots containing 50 g of soil.

The PHB polymers as sliced at the final concentration of 1% (low dose, L) and 2% (High

dose, H) were added to the soil. The following treatments were planned:

1. 50 g soil + 0.5g PHB (L treatment)

2. 50 g soil +1g PHB (H treatment)

3. untreated soil (B treatment)

The pots were incubated under controlled temperature, light and humidity. The moisture

content of each soil–biomaterial mixture was adjusted to 50% of water holding capacity. The

monitoring of the microscale experiment consisted of soil sampling carried out at one, three

and four weeks from the experiment set up.

3.5.3. Methods

3.5.3.1. Chemical Parameters (pH, electrical conductivity, total organic carbon, total

nitrogen, water soluble carbon, nitrate, ammonia, total and available elements)

Electrical conductivity (E.C.) and pH were measured in 1:10 (w/v) aqueous solution. Total

organic carbon (TOC) and total nitrogen (TN) content of soil were determined by RC-412

multiphase carbon and FP-528 protein/nitrogen determinator (Leco, USA), respectively.

Ammonium and nitrate were determined with selective electrodes. Water Soluble Carbon

(WSC) was determined spectrophotometrically at 590 nm by using the method of dichromate


- 98 -

oxidation (Garcia, et al. 1990) . Total and available elements were determined with Atomic

Absorption Spectrometry (AAS). Total and available phosphorus were determined by the

Murphy and Riley (1962) method

3.5.3.2. Biochemical Parameters

Protease activity

The proteases are a group of hydrolytic enzymes connected to the nitrogen cycle; they

catalyzed the hydrolysis of proteins in oligopeptides or dipeptides. The ammonium released

by the hydrolytic reactions was measured by an ammonium selective electrode (Bonmatı, et

al.2009).

Tests: 0.5g of soil with 2ml of phosphate buffer 0.1 M at pH 7 and 0.5ml of substrate BAA

(N- -Benzoil-L- -Arginammide-Hydrochloride) 0,03M.

Controls: 0.5g of soil with 2ml of phosphate buffer 0.1 M at pH 7.

Tests and controls were shacked in a thermostatic bath at 37°C for 1 hour and half. Then the

samples were brought to a final volume of 10 ml with bi-distilled water and centrifuged for

10 minutes at 3500 rpm. The supernatant was read with a NH3 electrode. The results are

expressed in mgNH3 kg-1

h-1

.

Phosphatase activity

The phosphatase catalyzes the hydrolysis of phosphoric ester to phosphate. The methodology

consists in the determination of the release of para-nitrophenol (PNF) from the hydrolysis of

the para nitrophenyl-phosphate- hexahydrate (PNP) (Tabatabai and Bremner 1969).

Tests: 0.25g of soil + 2ml of maleate buffer 0.1 M at pH 6.5 + 0.5ml of PNP substrate 0.12

M.

Controls: 0.25g of soil + 2ml of maleate buffer 0.1 M at pH 6.5.

Tests and controls were shacked in a thermostatic bath at 37°C for 1 hour and half. 0.5 ml of

substrate (PNP) was added also to the controls and they were made cold at 4°C for 10

minutes, to block the reaction. Then 0,5ml of CaCl2 0.5M and 2ml of NaOH 0.5M were

added and the samples were brought to a final volume of 10 ml with bi-distilled water.

Samples were centrifuged for 10 minutes at 3500 rpm. The supernatant was read with the

spectrophotometer at a wave length of 398 nm. The optic densities picked up by the

instrument are transformed in concentrations referred to a standard straight line obtained by

the known concentration of PNF. The results are expressed in mgPNF kg-1

h-1

.

Dehydrogenase activity

Dehydrogenase enzymes catalyze the oxidation of organic compounds. The reaction

substrate consist in the organic substance, while the synthetic cofactor utilized for the

measure of the dehydrogenase activity consists in the INT (P-Iodio-Nitro-Tetrazolium-

chloride) which makes a chlorate product through reduction, INTF (p-Iodo-Nitro-

Tetrazolium-Formazano) determinable with a spectrophotometric methodology. This

enzymatic activity was determined using the methodology studied by Masciandaro et al.

(2000).

Tests: 0.1g of soil with 0.2ml of INT substrate at 0.5% (in bi-distilled water) and 0.1ml of bi-

distilled water (to bring the sample at the 60% of the field capacity).

Controls: 0,1g of soil with 0,3ml of bi-distilled water (to bring the sample at the 60% of the

field capacity).

Tests and controls were let rest for 20 hours in dark place; the tubes were not covered

because the INT prevails oxygen (the natural substrate of the dehydrogenase) as electrons

acceptor. The INTF is the product of the oxido-reduction and it is insoluble in water; it was

extracted through the addition of an extractant solution (tetrachloroethylene/acetone, 1/1.5).

Experiment

- 99 -

The solution was vortexed for one minute and centrifuged at 3500 rpm for 10 minutes. The

supernatant was used for the spectrophotometric measure, using a wave length of 490 nm,

referring it to the control. The optic densities picked up by the instrument are transformed in

concentrations, express in mgINTF kg-1

h-1

, referred to a standard straight line obtained by

the known concentration of INTF.

Arylesterase activity

Arylesterase activity has been measured using p-nitrophenyl acetate (p-NPA) as substrate

and dimethylsulfoxide as solvent, modified universal buffer at pH 7.5. The product of the

reaction (p-nitrophenol), after an incubation at 55 °C for one hour, has been tested

spectrophotometrically at 298 nm.

PHB-depolymerase activity

PHB depolymerase activity assay was carried out at 37 °C in 0.1 M potassium phosphate

buffer (pH 7.4). The amount of 3-hydroxybutyrate (3HB) liberated from the P(3HB)

poliymer during the enzymatic hydrolysis was determined by the ultraviolet method (Abe et

al. 2010; Doi et al. 1994). Impurities with absorption at 210 nm decrease the sensitivity of

the assay.

3.5.3.3. Biological parameters

Colony forming units (CFU) were determined in aqueous solutions of soil (1:10 (w/v) soil:

water solutions) based on the surface-plate counting proce-dure (Jayasekara et al., 1998). The

extraction was carried out in tubes with sterile water and vortexing for 2 min at room

temperature. Serial dilutions were made for the extracts using distilled water. A volume of

0.2 ml was spread onto plates prepared with adequate growing medium: plate count agar

(Sigma Aldrich) for total cultivable microbial population and Pseudomonas agar base (Sigma

Aldrich) for Pseudomonas genus. Plates were incubated at 29 °C for 48 and 72 h for plate

count agar and Pseudomonas agar base, respectively. The CFU per gram of soil were

calculated (Picci and Nannipieri, 2002).

3.5.3.4. Germination and Growth Test

Germination and Growth test was done on the different soil treatments to evaluate the

seeding and growth capability of the plants Avena sativa and Raphanus sativus. For each

treatment, a pot sowed with 100 seeds of Avena sativa and a pot sowed with 100 seeds of

Raphanus sativus at a depth of 3 cm were carried out.

Plants‘ growth was measured after about 10 days.

The Germination and Growth Index (GI%) was calculated by using the following equation:

GI (%) = x x 100

3.5.4. Statistical Analisys

The STATISTICA 6.0 software (StatSoft Inc., Tulsa, Oklahoma, USA) was used for all

statistical analysis. All numerical parameters before statistical analysis were normalized and

autoscaled: the result for each variable is a zero mean and an unit standard deviation.

n. seed germinated test

n. seed germinated control

length plant test

length plant control


- 100 -

PCA is a multivariate statistical data analysis technique which reduces a set of raw data into

a number of principal components which retain the most of the variance within the original

data in order to identify possible patterns or clusters between treatments and variables

(Carroll, et al. 2004). The PCs were extracted by applying the principal of main axis method.

Only component loadings >0.7 were considered for interpretation of the PCs. In PCA

analysis, the data are decomposed into separate sets of scores and loadings for each of the

two modes of interest (treatments and variables) and the whole variability of the data is

explained in order to provide a clear and more interpretable visualization of data structure in

a reduced dimension. In addition, PCA analysis gives information that can also be clearly

presented graphically.

Result and Discussion

- 101 -

4. RESULTS AND DISCUSSION

As has been mentioned in previous chapter one of the aim of the present Doctoral Thesis is

to identify the influence of particular and innovative additive as it is Joncryl ADR-4368-CS

and hazelnuts shell powder (here labeled as Lignin) on the biodegradation (mineralization) of

binary and ternary composites in soil medium, mature compost and river water. In particular,

it has been also studied the concentration of Joncryl (J) and Lignin (LN) and the influence of

this parameter on the biodegradation propensity of the binary system.

It is well known that the main factors that affect the biodegradation behavior are the

chemical composition of the material, molecular weights, the chemical bonds in the structure,

the crystallinity of the material, the nature of the surfaces, the indigenous micro-flora and the

environmental conditions (Lutz and Falcao 2011). Then in order to explain the results

obtained from the biodegradation tests in soil, mature compost and river water, it is

important to assess the thermal properties of PHBV and PLA based composites by using

differential scanning calorimetric (DSC) and thermogravimetric analysis (TGA), the

structural analysis of the raw material and of binary composites by FT-IR, as well as, the

determination of the molecular weight for the raw materials and composites by GPC.

Moreover, the evaluation of the thermal properties of the composites prepared, together with

the results of the biodegradation experiments and their processability characteristic can be

useful for envisaging future applications and end life disposal strategies for the obtained

composites.

We start the present chapter by analyzing the results obtained from DSC and TGA thermal

characterization for PHBV and PLA based composites, followed by some of the

characterization done for PLA and PHBV through GPC analysis and FT-IR analysis. The last

part will deal with the discussion of the results related with the processability characteristics

of the raw materials and their composites in melt mixing discontinous experiments

(Brabender). And we will end with the biodegradation experiments in soil, mature compost

and river water

.

4.1. Differential Scanning Calorimetry (DSC)

4.1.1. PHBV Based Blends and Composites

From the DSC experimental data, it is possible to collect information related with the thermal

transition that characterizes amorphous and semi-crystalline polymers. The thermal

transitions are: melting temperature, (Tm); glass transition temperature, (Tg) and

crystallization temperature (Tc). In general, these properties are obtained from the second

heating run and the cooling run of each sample. An adequate experimental set-up for the

thermal analysis experiment is important to do with the aim to identify the real

thermodynamic behavior of the materials without the influence of their previous thermal

history. Another important parameters, also obtained from DSC thermal analysis, are the

enthalpy of fusion ( Hm) and crystallization ( Hc). In particular, the crystallinity (Xc %) of

the raw materials as well as the final composites is determined by considering the enthalpy of

fusion during the second heating run.

The crystallinity determination takes into account corrections related with the weight fraction

of the semi-crystalline component in the blend, the presence or not of the cold crystallization


- 102 -

(Tcc), and the theoretical enthalpy of the polymer used considering a crystallinity of 100%

(Wang et al. 2010, Avella et al. 2000, Sarasua et al.2005).

In the present part emphasis will be done in the correlation between the crystallinity degree

of the material and the composites prepared and its influence on the biodegradation

behaviour results. Additionally, the thermal properties will be analyzed in order to analyze

the influence of Joncryl and Lignin on the melting behaviour of the binary blends.

Table 4.1 shows the thermal properties of PHBV raw material and its based composites. The

influence of the concentration of Joncryl (J), Lignin (LN) and Montmorrillonite (Dellite – D)

have been also evaluated.

Table 4.1. Thermodynamic Parameters for PHBV Based Blends and Composites: Tm,

Tc, Tg and Xc.

Sample Tg

(°C)

Tcc

(°C) Hcc

(Jg-1

)

Tc

(°C) Hc

(Jg-1

)

Tm

(°C)

Xc§

(%) Hm

(Jg-1

)

PHBV - - - 84 103 170 80 117.0

PHBV + J0.2 - - - 96 91.4 165 73 106.6

PHBV + J5 2 47 6.0 79 65.3 163 65 96.3

PHBV + LN10 - - - 89 50.1 178 46 61.1

PHBV + LN20 - - - 91 63.4 174 69 80.5

PHBV + LN10 + J5 - - - 87 48.6 176 50 62.0

PHBV + LN20 + J5 - - - 89 44.7 183 51 56.1

PHBV + D3 - - - 88 64.8 171 55 78.4

PHBV + D6 - - - 88 56.5 170 49 67.3

PHBV + D3 + J2 - - - 73 77.7 160 73 100.9

PHBV + LN10 + D3 - - - 92 61.2 175 56 71.4

PHBV + LN20 + D3 - - - 91 54.7 178 50 56.2

PHBV + LN10 + D3 +

J2

- - - 85 52.8 177 56 69.8

PHBV + LN20 + D3 +

J2

- - - 88 37.2 179 43 47.2

PHBV + LNF10* -15 39 48.7 37 7.8 128 9 60.0

§ Xc =[(ΔHm –ΔHcc)/ΔHm100% x ]100 for samples with cold crystallization, Xc =

(ΔHm/ΔHm100% x )100 where ΔHm PHB100% crystalline= 146 Jg-1 (Wang et al.

2010) and is the weight fraction of PHB in the composite

* Sample with multiple melting point: Tm1 = 63 °C, Tm2 = 88 °C

From DSC results, lignin and inorganic component (Montmorrillonite - Dellite) decreases

the enthalpy changes (melting and crystallization) of composites compared with the raw

material (PHBV), a little amount of Joncryl (0.2%) has no effect on the enthalpy changes,

however 5% of its addition can also decrease the melting enthalpy as well as the

crystallization. In particular, the addition of 5% by weight of Joncryl produces on PHBV

matrix hindering effects on the crystallization ability of the PHBV polymer chain during the

cooling step and, as a consequence, a cold crystallization is observed during the second

heating.

Meanwhile, the addition of Joncryl and Dellite can decrease or maintain the melting

temperature of PHBV matrix, contrary, the lignin addition increases the PHBV melting

temperature from 4 until 8 degrees. The higher melting temperature value was obtained for

the sample named PHBV + LN20 +J5 having a melting peak at 183 °C, comparing this value

with the raw material, an increment of 13 °C was observed. The higher the melting


- 103 -

temperature of the final composite the higher will be the extension of the degradation during

an eventually second processing step. This statement is correlated with the fact that the

decomposition temperature of PHBV in the melt (140 - 170° C) is closer to the melting

temperature of the polymer (Arrifin et al. 2008, Arrifin et al. 2009).

All the composites (the inorganic and organic ones) prepared, posses a lower crystallinity

degree than the raw material (PHBV). For the binary systems the lower crystallinity degree

was obtained for the sample PHBV + LN10 with 46%, followed by the sample PHB + D6

having 49%. Instead the samples with the higher crystallinity degree are PHBV + J0.2 and

PHBV + D3 + J2, both with 73%. Nevertheless, a dramatic change in the crystallinity has

been found for the sample PHBV + LNF10 (with around 9%), this sample is characterized by

the presence of a functionalized lignin in a concentration of 10% by weight. The

functionalization of the lignin was done with the aim to improve its compatibility with the

hydrophobic PHBV matrix. The chemical components for the functionalization were stearic

acid in the presence of a catalyst. The conditions of the reaction as well as the analysis of the

functionalized lignin are out of the scope of the present doctoral thesis work.

The thermogram traces for the chain extender additive (Joncryl) are shown in the Figure 4.1.

It is possible to observe a double melting peak for the pure PHBV and by increasing the

amount of Joncryl in the blend the presence of a glass transition (Tg) temperature and a cold

crystallization (Tcc) are evidenced together with the disappearance of the first PHBV‘s

melting peak.

Figure 4.1. Thermogram Traces for PHBV Blended with Joncryl Additive at Different

Concentration, 0%, 0.2% and 5%.

Figure 4.2 shows the thermograms traces for the organic composites (PHBV + LN10 and

PHBV + LN20). Adding hazelnut shell powder in the composite, was observed an increment

in the melting temperature peaks of pure PHBV. In particular, with an higher concentration

of lignin an inversion in the heat flow was observed for the melting peaks of the sample. In

both composites the melting peaks are overlapped compared with the pure PHBV.


- 104 -

Figure 4.2. Thermogram Traces for PHBV Blended with Hazelnut Shell Powder (Lignin) at

Different Concentration, 0%, 10% and 20%.

Figure 4.3, shows the thermogram traces related with the addition of organic filler as

hazelnut shells powder (lignin) with different chemical treatment (original and functionalized

lignin at 10% by weight). Functionalized treatment on the lignin showed clearly more

significant effect on DSC traces compared with the unmodified lignin. The presence of a Tg,

a Tcc as well as multiple melting peaks suggest the presence of different PHBV crystal

population, that can be originated from the chemical and/or physical interaction between the

pure PHBV and the functional groups inserted in the lignin during the processing conditions.

Figure 4.3. Thermograms Traces of PHBV Based Composites with Different Chemical

Treatment: Original Lignin and Functionalized Lignin Powder.

Increasing the level of complexity by adding both Joncryl and lignin, in different weight

percentage, an interesting phenomenon was observed in Figure 4.4. The effect of the lignin

on the PHBV composite thermal transitions is more pronounced than the effect of the Joncryl

(at 5%). So it indicates that the lignin influence on the heat flow, in the analysis by DSC, is

more obviously than the Joncryl. In particular, the Tg and the Tcc (produced by adding 5% of

Joncryl) were completely disappears in the PHBV + LN20 + J5 composite.


- 105 -

Figure 4.4. Thermograms Traces of PHBV Based Composites with Different Lignin

Concentration: 10% and 20%, and with 5% of Joncryl.

The effects of addition of inorganic fillers as Montmorillonite (Dellite) on DSC behaviour

are shown in Figure 4.5. By doing a comparison between the organic and the inorganic filler

on their role in the final composite thermal behaviour one can draw conclusions as discussed

before. The overlapping in the PHBV melting peaks was observed for all the binary and

ternary systems and an increment in the higher melting peak was also observed.

Figure 4.5. Thermograms Traces of PHBV Based Composites with Different Concentrations

of Lignin (10% and 20%), Inorganic Filler as Montmorillonite (Dellite) (3% and 6%). PHBV

and its Binary Blends.

Final comparison for the blends containing inorganic fillers as Montmorillonite (Dellite) and

lignin at different percentage (20% and 10%), were also determined and analyzed by DSC

and the results are shown in Figure 4.6. From the DSC traces similar conclusions were

obtained when compared with the results of previously discussed (see values on Table 4.1).

The addition of 3% of Dellite and 2% of Joncryl in the PHBV, was not producing changes on

the PHBV thermal behaviour. Additionally, the ternary composites still shown the same

trend already discussed, that is: the higher the amount of lignin in the composite the higher is

its effect on the PHBV heat flow changes and this effect is similar to that observed in the

binary composite PHBV + LN.


- 106 -

Figure 4.6. Thermograms Traces of PHBV Based Composites with Different Concentrations

of Lignin (10% and 20%), Inorganic Filler as Montmorillonite (Dellite) (3%), Joncryl (2%).

PHB and its Ternary Blends.

4.1.2. PLA Based Blends & Composites

With the aim of assessing the influence of the chain extender additive (Joncryl) and the

organic filler (lignin) on the thermal properties of poly(hydroxyalcanoate) PHA, PLA based

composites characterized by similar compositions, in terms of additives, as PHB based

composites have been prepared.

The thermal properties of the new composites are shown in Table 4.2. The presence of

Joncryl and lignin in PLA based composites do not produce appreciable changes in the PLA

thermal properties. The glass transition temperature and the cold crystallization temperature

remain almost unchanged, as well as, the melting temperature. However, occurring as in the

case of PHBV, higher amount of Joncryl (5%) in PLA blends decreases the melting

temperature of PLA by 6 ℃.

Table 4.2. Thermodinamic Parameters for PLA based Blends and Composites: Tm, Tc,

Tg and Xc.

Sample Tg

(°C)

Tcc

(°C) Hcc

(Jg-1

)

Tc

(°C) Hc

(Jg-1

)

Tm

(°C)

Xc§

(%) Hm

(Jg-1

)

PLA 60 113 30.8 96 0.9 156 7.5 37.7

PLA + J0.2 61 115 31.6 75 0.5 153 0.5 32.1

PLA + J5 60 123 24.0 75 0.5 150 0.8 24.7

PLA + LN10 60 110 31.1 80 1.0 156 1.4 32.3

PLA + LN20 60 113 25.9 77 0.7 150 2.9 28.1

PLA + LN10 + J5 60 125 18.2 74 0.8 152 5.1 22.2

PLA + LN20 + J5 61 125 13.2 76 0.4 155 1.6 14.3

§ Xc =[(ΔHm –ΔHcc)/ΔHm100% x ]100 for samples with cold crystallization, Xc =

(ΔHm/ΔHm100% x )100 where ΔHm PLA 100% crystalline= 93 Jg-1 (Sarasua et al.

2005) and is the weight fraction of PLA in the composite.


- 107 -

The major influence found for both additives (Joncryl and lignin) was on the PLA

crystallinity degree. Normalizing the crystallinity degree value by dividing it by the Xc value

of the pure polymer, it was found that both Joncryl and lignin reduce dramatically the PLA

crystallinity degree when their effect on PHBV (Table 4.3). In particular, the major

modification was obtained for Joncryl at low concentration (0.2%). This result suggests that

the chain extension reaction, promoted by the epoxides functional groups contained in the

Joncryl chemical structure, has been more favored in the PLA than in PHBV. Probably the

concentrations of reactive functional groups in the PLA main chain and in the end-chain are

higher than in the case of PHBV. Similar behavior was also found for the effect of lignin in

the PLA based composites.

Molecular weight determinations have been done in order to clarify the above mentioned

hypothesis. The results are shown in Table 4.4.

PLA sample, containing 0.2% by wt. of Joncryl shows the higher variation in the molecular

weight (Mw) and in the polydispersity index (P.I) compared with the pure PLA. This result is

in agreement with the crystallinity degree variations observed for the PLA + J0.2% sample

(see Table 4.3). Probably, the lower concentration of chain extender additive favored its

dispersion inside the melted polymer and its compatibility with the PLA. Thus, the reactive

conditions are favored and, in this way, the chain extension reaction is effective. It is

expected that an increment in the molecular weight weight, through chain extension reaction

with chemically different reagent, and the formation of branching can reduce the crystallinity

of the neat PLA. The presence of lignin in the PLA and in the samples containing Joncryl at

5% did not produce significant variations in the cristallinity values.

Unfortunately, molecular weight determinations for the PHBV samples and composites were

difficult to perform due to the fact that the final samples were all insoluble in chloroform.

The insolubility can be due to the high crystallinity of the PHBV before and after blending or

by the formation of crosslinking between polymer chains thus hindering their solubility.

PHBV has higher molecular weight than PLA (see Table 4.4), however its P.I is slightly

lower. This fact is in agreement with the above mentioned tendency related with the

effectiveness of Joncryl on PLA matrix instead of PHBV matrix.

Figure 4.7 is showing the general reaction mechanism between Joncryl as reactive additive

and the functional groups present in the melt.

Figure 4.7. General Mechanism of Chain Extension Reaction Between Polymers and Joncryl

Additive (Arrifin et al. 2008, Arrifin et al. 2009, Pilla et al. 2009).


- 108 -

Table 4.3. Normalized Crystallinity Degree for PLA and PHBV Based Blends and

Composites.

Component XcPHA / XcComposite

PLA PHB

J0.2 15.0 1.1

J5 9.4 1.2

LN10 5.4 1.7

LN20 2.6 1.2

LN10 + J5 1.5 1.6

LN20 + J5 4.7 1.6

Table 4.4. Molecular Weight Values for PHBV and PLA Blends & Composites.

Sample Molecular weight by GPC

Mw Mn P.I

PHBV 482.620 311.800 1.5

PLA 163.390 89.193 1.8

PLA + J0.2 208.500 90.213 2.3

PLA + J5 233.000 148.720 1.6

PLA + LN10 152.200 100.900 1.5

PLA + LN20 163.000 105.220 1.5

PLA + LN10 + J5 238.630 141.880 1.7

PLA + LN20 + J5 226.520 144.950 1.6

In the case of PLA samples with Joncryl additive at 0.2 and 5 % by weight, the results are

shown in Figure 4.7. Similar results can be obtained by comparing it with the PHBV thermal

behavior. The addition of Joncryl can significant modify the heat flow trace, higher is the

percentage of additive was added, higher is the heat flow variation is observed.

Figure 4.8. Thermograms Traces of PLA Samples at Different Concentration of Joncryl: 0%,

0.2% and 5%.

Figure 4.8 shows the thermogram traces recorded for PLA based composites with 10% and

20% of lignin from hazelnut shell.

PLA thermogram (Figure 4.9) shows an endothermic peak connected to the isothermal

crystallization (T = 110°C) of this material that, having an high glass transition temperature


- 109 -

compared to the ambient temperature, shows an amorphous part also in the melt state of the

polymer (Day 2006). Not significant variation in the heat flow traces were observed by

varying the amount of lignin in the composite. This observation is in agreement with the

obtained DSC analytical results (see Table 4.2).

The Joncryl addition makes more distinct the melting peak at 160°C and the shoulder at

154°C observed in the neat PLA thermogram. The double melting peak observed for

PLA/Joncryl blends can be connected to a different morphology of the crystalline phase

(width of the lamellar structure and spherulites dimensions), obtained by crystallization in

the melt and isothermal crystallization (Pilla et al.2009).

A chain extender concentration above 2% produces an increment for the isothermal

temperature about of 10°C for PLAJ5 (Tcc= 127°C) composite compared to that of neat PLA

(Tcc= 118°C), thus the presence of a branched structure makes slow the crystallization

process

Joncryl addition until .2%, the inorganic component and the lignin (10%) increase the

crystallinity degree for the composites compared to that of neat PLA (Xc = 43.1), whereas

the lower values have been recorded for PLAJ5, PLALN20J5, PLALN20D3, PLALN20D3J5

composites. This trend can be explained considering an increment for the composites

molecular weight (Perego et al. 2006), as detected by the GPC analysis.

The high value for the glass transition temperature (Tg=60°C) confirms the rigidity for the

polymeric material and its composites

The lignin addition (10% and 20%) can be underlined by the presence of an exothermic peak

near to the endothermic peak of the polymer, well pronunced and visible in the respective

thermograms. This phenomemenon is connected with PLA isothermal crystallization, that

occurs during the heating process (Li et al. 2003).

Increasing ligni amount until to 40%, a decrease for the melting temperature from 169.9°C to

161.8°C and for the glass transition temperature from 63.4°C to 57.3°C, was observed.

This trend can be explained, considering the mobility of the _OH phenolic groups, able to

create strong intermolecular hydrogen bonds with the PLA carbonilic groups (Li et al. 2003).

Figure 4.9. Thermograms Traces of PLA Composites at Different Concentration of Lignin:

0%, 10% and 20%.

In the case of PLA composite, which the results are shown in Figure 4.10, was concluded

that the addition of both additives lignin and Joncryl in different percentage seems not

originated significant changes in the DSC thermograms. However, by increasing the

percentage of lignin at 20% and Joncryl at 5%, a notorious modification on the heat flow

traces was observed. In particular, these changes can be attributed to the presence of Joncryl


- 110 -

instead of lignin. Contrary to PLA, the PHBV matrix has been affected either by Joncryl as

by lignin.

Figure 4.10. Thermograms Traces for PLA Based Composites Having Organic Filler as

Hazelnut Shells Powder (Lignin) at Different Concentration: 10% and 20%, and with

Additive as Joncryl at 5% by Weight.

4.2 ThermoGravimetric Analysis (TGA)

4.2.1. PHBV Based Blends & Composites

The onset temperature (Ton

) of decomposition was determined as the temperature

corresponding to the crossover of tangents drawn on both sides of the thermogravimetric

trace. The residue was evaluated as the residual weight at the end of the experiment generally

stopped at 700°C. The main results of thermal characterization-TGA data of PHBV based

composites with respect to thermal properties as thermal stability (Ton) and the residue (%) at

700°C are reported in Table 4.5.


- 111 -

Table 4.5. Thermal Properties for PHBV Based Blends and Composites as recorded

Under Nitrogen Atmosphere.

Sample Codes TGA N2 DTGA

Tonset

a)

(°C)

Residueb)

(%)

Tp1

(°C)

Tp2

(°C)

Tp3

(°C)

PHBV 250 0.54 274 - -

Joncryl (J) 241 0.80 294 349 402

Lignina (LN) 260 22.6 286 360 431

Montmorillonite (Dellite D) 251 62.4 321 407 589

PHBV + J0.2 256 0.40 279 - -

PHBV + J2 249 0.39 274 375 -

PHBV + J5 252 0.58 265 399 -

PHBV + LN10 257 2.62 278 352 -

PHBV + LN20 252 3.81 272 348 -

PHBV + LN10 + J5 269 2.67 284 362 406

PHBV + LN20 + J5 269 3.50 282 361 410

PHBV + D3 261 2.32 283 - -

PHBV + D6 227 4.82 266 463 -

PHBV + D3 + J2 260 2.38 286 384 -

PHBV + LN10 + D3 268 4.00 287 367 -

PHBV + LN20 + D3 264 5.61 284 367 -

PHBV + LN10 + D3 + J2 264 3.42 286 371 -

PHBV + LN20 + D3 + J2 271 4.78 285 372 -

PHBv + LNF10 215 2.54 243 311 -

a) At 10%-wt loss b) Residue at 700°C

TGA data for PHBV composites (Table 4.5) indicated that lignin and inorganic filler can

significantly increase the onset temperature and residue for PHBV composites. These

additives increased the thermo stability of the materials, meanwhile, Joncryl‗s addition has

not significant effect on the above parameters. From the results, addition of 3% of Dellite

shows better thermo-stability compared with the addition of 6%.

The organic composites PHBV + LN, PHBV + LN + J, the inorganic (PHBV + D) ones and

hybrid ones, PHBV + LN + D, PHBV + LN + D + J, based on PHBV were investigated in

respect of the thermal properties by TGA (Table 4.5) with the aim to obtain useful

information on better suited processing temperatures to be applied eventually by the

converters.

TGA (a) and DTGA (b) traces for PHBV based composites containing the chain extender (J)

are reported in Figure 4.11a-b) whereas those relevant to the lignin, alone or in combination

with Joncryl are reported in Figure 4.12a-d). The Joncryl dispersion (0.2%, 2%, 5%) in the

polymer matrix produces a light thermal destabilization at Joncryl concentrations of 2%, as

observed by the drop of the onset temperature.


- 112 -

The blend containing Joncryl (0.2% - wt) is the only one that presents a higher thermal

stability with respect to PHBV processed in Brabender.

Figure 4.11. TGA (a) e DTGA (b) Traces Recorded for PHBV/Joncryl Blends.

DTGA traces (b) shows only one degradation peak for all PHBV based blends containing

Joncryl with a shoulder at 375°C for the composite PHBV/J2 and at 399°C for the blend

PHBV/J5.

The lignin addition to PHBV produces an increase of 20°C for the thermal stability of

PHBV/LN/J composites respect to neat PHBV and the binary system PHBV/Lignin, as

observed by the registered Ton values (Table 4.5).

This trend was confirmed also by the residue value (3.5%) as determined at 700°C. The

recorded higher data can be correlated to the minor propensity towards degradative

reactions for the material composites.

The ternary systems PHBV/LN/J show a multiple trend for the DTGA trace, that has

three degradation peaks connected to the major material weight losses.

Also the ternary systems PHBV/LN/D and quaternary ones PHBV/LN/D/J shows Ton

values by 20°C higher than PHBV processed in Brabender. The derivative trend has a

simple profile as noticed from the loss of one degradation peak. The chain extension

reaction promoted by Joncryl can stabilize the quaternary systems making possible their

thermal degradation with a minor number of weight losses (figure 4.13).


- 113 -

Figure 4.12. TGA (a, c) e DTGA (b, d) Traces Recorded for PHBV/Lignin and

PHBV/Lignin/Joncryl Composites.

Figure 4.13. TGA (a, c) e DTGA (b, d) Traces Recorded for PHBV/Lignin/Dellite and

PHBV/Lignin/Dellite/Joncryl Hybrid Composites.

PHBV/D6 composite is characterized by a lower onset temperature (Ton = 227°C) compared

to PHBV/D3 composite (Ton = 261°C) which posses higher thermal stability than the pure

PHBV (Ton = 250°C).


- 114 -

Figure 4.14 shows a comparison between the TGA (a) and DTGA (b) traces for PHBV

based composites loaded with lignin and functionalized lignin with stearate groups, at

content of 10%-wt of lignin.

Figure 4.14. TGA (a) e DTGA (b) Traces of PHBV/LN10 and PHBV/LNF10 Composites.

The introduction of functionalized lignin results in about 35°C decrease with respect to

PHBV and PHBV/LN10 composite. The thermal degradation for PHBV/LNF10 composite

starts at lower temperatures connected to a derivative shift toward temperatures lower than

200°C.

4.2.2. PLA Based Blends & Composites

Thermogravimetric analysis, under air atmosphere, was performed on native PLA and on

PLA based blends and composites loaded with Joncryl, lignin, alone or in combination.

Figure 4.15 shows TGA (a) and DTGA (b) traces for these blends and composites; also the

thermal decomposition profile for lignin and Joncryl was recorded to make a comparison

between the single components and their synergistic effect inside the composite.

The chain extender (Joncryl) has a minor thermal stability than pure PLA, as observed by the

lower Ton value (Table 4.6).

The first degradation peak, present in the Joncryl DTGA profile, showed three shoulders,

that derive from the overlapping between the main peak and smaller peaks located at

temperature of 269°C, 293°C, 314°C. These thermal effects can be correlated with the

formation of low molecular weight components or ring opening reactions for the epoxy

groups.

Lignin powder has a TGA trace (Fig. 4.15a), placed in a temperature range larger than the

same recorded for the selected blends and composites.

Its onset temperature is 261°C, whereas the residue recorded at 700°C is around at 1.15%,

that is an high value connected with the presence of aromatic rings, thermally stable, in the

lignin structure.

The peaks molteplicity, present in the DTGA trace (Fig. 4.15b), was an experimental

evidence of the ligno-cellulosic material composition. In fact the organic filler derives from

the grinding of the hazelnut shells and from sieving its powder: so lignin is not the only

component of this material, because of notable amount of cellulose and hemicellulose are

contained onto the material.


- 115 -

(a) (b)

Figura 4.15.TGA (a) e DTGA (b) Traces of PLA/Lignin, PLA/Joncryl Blends and Hybrid

Composites (PLA/Lignin/Joncryl) Under Air Atmosphere.

The onset temperature represents the starting for the composite degradation and it is

connected with the thermal stability of the material. This parameter gives suitable

information on the polymeric materials processing temperature and features.

The Joncryl addition (5 wt-%) alone or in combination with the organic filler (lignin) showed

a decrease of about 30°C for the PLAJ5 blend and for PLA/LN20/J5 composite thermal

stability, while PLA/LN20 blend and PLA/LN10/J5 composite showed an increment of

about 10°C.

The residue consists in the material amount that remains uncombusted at the end temperature

of the experiment. So it is quite high for all the analyzed blends and composites that contain

aromatic and epoxy rings for which, high temperature are needed to be utilized for the

starting of thermal degradation.

Thermal properties are collected in Table 4.6.

Table 4.6. Thermal Properties of PLA Based Blends & Composites Under Air Atmosphere.

Samples TGA air DTGA

Tonset

(°C)

Residue

(%)

Tp1

(°C)

Tp2

(°C)

Tp3

(°C)

Tp4

(°C)

PLA 309 0.23 359 402 - -

Lignin 261 1.15 292 323 390 452

Joncryl 254 0.18 328 345 416 499

PLA + J0.2 326 0.45 302 365 429 460

PLA + J5 284 0.23 306 323 336 409

PLA + LN10 295 0.47 337 399 441 -

PLA + LN20 315 0.39 276 362 461 -

PLA + LN10 + J5 319 0.30 334 366 482 505

PLA + LN20 + J5 295 0.57 290 357 461 495


- 116 -

Figure 4.16 shows the Joncryl concentration effect on the PLA thermal degradation profile.

(a) (b)

Figura 4.16. TGA (a) and DTGA (b) Traces for PLA/Joncryl Blends Under Air Atmosphere.

A small concentration of chain extender does not modify the shape of PLA thermal

degradation profile (Fig. 4.16a) and the temperature range (260-330°C) in which, the onset

temperature is comprised. Increasing the concentration until to 5%, a more complex profile

with a degradation peaks molteplicity (Fig. 4.16b) was produced and it connected with

oxidative reactions and cross-linking that occur between the polymer and the chain extender.

Table 4.7 collects the data related to PLA based blends and composites thermal properties

obtained for TGA analysis under nitrogen atmosphere in order to make a comparison

between the blend or composite behavior and the component effect under the two different

atmospheres.

Table 4.7. Thermal Properties for PLA Based Blends & Composites Under Nitrogen

Atmosphere.

Samples

TGA

DTGA

Tonset

(°C)

Residue

(%)

Tp1

(°C)

Tp2

(°C)

Tp3

(°C)

Tp4

(°C)

PLA 313 0.91 359 441 - -

Lignin 260 22.5 286 360 431 -

Joncryl 240 0.86 294 320 350 402

PLA + J0.2 313 1.19 300 356 - -

PLA + J5 276 0.94 317 399 470 -

PLA + LN10 266 4.61 265 309 460 -

PLA + LN20 276 5.01 258 313 775 -

PLA + LN10 + J5 271 3.95 271 319 402 462

PLA + LN20 + J5 291 7.94 278 324 375 439


- 117 -

Figure 4.17 reports TGA (a) and DTGA (b) for Pure PLA and Its Composites, Recorded

under Nitrogen Atmosphere.

(a) (b)

Figura 4.17. TGA (a) e DTGA (b) Traces of PLA/Lignin, PLA/Joncryl Blends and Hybrid

Composites (PLA/Lignin/Joncryl) Under Nitrogen Atmosphere.

In nitrogen atmosphere, the addition of the organic filler (lignin), the chain extender (Joncryl)

and their combination loaded inside the composite with the neat PLA, produces for the

blends and composites a lower thermal stability than the native PLA, as observed by the Ton

values (Table 4.7).

DTGA profile (Fig. 4.17b) is very complex (presence of four peaks) for Joncryl and for the

based PLA composites loaded with Joncryl and lignin. These peaks (Fig. 4.17b) are

comprised in the temperature range of 270-470°C and they are connected with the

degradation of low molecular weight component contained in the composites and with the

reactions on polymer and chain extender carbon backbone without taking into account the

oxidative phenomena that can occur between the composite components with the oxygen of

the air.

The residue value is higher than those recorded in air atmosphere for the same composites:

the Carbon residue remains in large amount after the burning of the sample.

The lignin powder shows a residue (22.5%) that can be correlated with the real lignin

fraction contained in the ligno-cellulosic material, because of the lignolitic part degrades

before 700°C (Brebu et al. 2010).

Figure 4.18 reports TGA (a) and DTGA (b) traces for PLA/Joncryl blends.

(a) (b)

Figura 4.18. TGA (a) and DTGA (b) Traces of PLA/Joncryl Blends Under Nitrogen

Atmosphere.

PLA/J5 blend showed a lower thermal stability (Ton = 276°C) than the pure PLA and

PLA/J0.2 blend (Ton = 313°C). So a small amount of chain extender does not affect the

thermal stability of the polymer. The additive results diluted in the polymer matrix and it


- 118 -

does not react with the end-groups of the polymer chains. Also the residues for PLA/J0.2

blend results rather high (1.19%).

4.3. Molecular Weight by Gel Permeation Cromatography (GPC) Analysis

The numeral (Mn) and the ponderal (Mw) molecular weight and the poly-dispersivity index

(Mn/Mw) were determined for PLA/Joncryl and PLA/Lignin blends (Table 4.8) with the aim

to investigate the organic filler and the chain extender concentration effect on the molecular

weight for PLA matrix and to obtain same informations related to possible reactions that can

be occurred between the polymer and these components.

Table 4.8. Numeral Molecular Weight (Mn), Ponderal Molecular Weight (Mw) and

Polidispersivity Index (Mn/Mw) for PLA/Joncryl and PLA/Lignin Based Blends.

Sample Mn

(kDa)

Mw

(kDa)

Mn/Mw

PLA 89 163 1.83

PLAJ0.2 90 208.5 2.31

PLAJ2 207 308 1.48

PLAJ5 149 233 1.56

PLALN10 101 152 1.51

PLALN20 105 163 1.55

PLALN10J5 142 239 1.68

PLALN20J5 145 226.5 1.56

The Joncryl loading inside the composite (Figure 4.19) caused a molecular weight increment

compared to neat PLA processed in Brabender, as observed by the lower retention times, at

which, the solutions containing Joncryl in concentration of 0.2%, 2%, 5%, eluted, producing

the resulting cromatograms.

The polidispersivity index showed a maximum value for the PLAJ0.2 blend and lower and

nearer values to PLA matrix (1.83) for PLAJ2 and PLAJ5 blends.

These blends have polymeric chains with different length and molecular weight that act as

functional groups, in major number compared to PLA polymer matrix.

Figure 4.19. Cromatograms for PLA and PLA/Joncryl Blends.


- 119 -

The lignin addition, alone or in combination with Joncryl did not create notable variations in

the polidispersivity index values, as observed from the overlapping and the same shape of

the recorded cromatograms (Figure 4.20).

(a) (b) Figura 4.20. Cromatograms for PLA, PLA/Lignin Blends (a) and PLA/Lignin/Joncryl

Composites (b).

4.4 PHAs Based Composites Processing Characteristic

The torque stability evaluation during the melt-blending process gives useful information

that can be correlated with the degradation of the material inside the Brabender mixer

chamber and with chemical reactions that can be occurred inside it such as cross linking and

chain extension. Likewise, the influence of the filler on the torque (plasticization) and the

induction time can be studied. In this way it is possible to suggest optimal processing

conditions for melt-processing blends and composites. The use of PHBV also has limitations.

One such limitation is its narrow processing window. The polymer is thermally unstable at

temperature above the melting point (around 170oC to 180

oC), and a drastic reduction in

molecular weight occurs during processing at temperatures in 180-200oC range. The

composites obtained by using inorganic and organic filler and innovative additive Joncryl

ADR-4368C results still brittles and present poor mechanical properties. It was difficult to

produce thin film by compression moulding for mechanical test, thus plasticization can be a

valid strategy to improve the final properties of the composites as well as their processability.

The other limitation is its relatively low impact resistance. In order to overcome these

shortcomings, efforts have been made to co-polymerize HB with other monomers HA, but

the cost is considerably high, and this is limiting its use. One possible approach to obtain a

less expensive thermoplastic polymer of improved mechanical properties is to blend PHBV

with another suitable polymer. Earlier studies have shown that blending polymers could lead

to improve properties, lower the cost and impart functionalities in blends (De Carvaho et al.

2008).

The presence of organic and inorganic fillers becomes an important parameters in the

assessment of the optimal processing conditions for the developed composite formulations.

Figures 4.20 - 4.21 shows the torque behaviour vs. time for the organic composites based on

lignin flour, obtained from the hazelnut shell grinding. Figure 4.20 reports on the profiles

obtained with lignin particle with size above 39 μm, whereas Figure 4.21 reports on the

profiles obtained with lignin particle size below 39 μm. Figure 4.22 reports on the traces

obtained for the torque behaviour recorded for the inorganic composites based on LDH and


- 120 -

Dellite at a load of 3 and 6wt-%.

Figure 4.23 shows the torque trend for the PHBV based samples and the additive Joncryl,

dispersed in the PHBV powder, at different concentrations (0.2, 2 and 5 weight percent).

Figures 4.24-4.25 show the torque trend for the hybrid composites, whereas the temperature,

inside the blending chamber (Brabender), and the final torque measures are listed in Table

4.8.

Figure 4.21. Torque Behavoiur vs. Time Recorded for Organic Composites Loaded with

Lignin (LN) with Particle Dimensions Up 39 Micron.

Figure 4.22. Torque Behaviour vs. Time Recorded for Organic Composites Loaded with

Lignin (LN) with Particle Dimensions Under 39 Micron.


- 121 -

Figure 4.23. Torque Behaviour vs. Time Recorded for the Inorganic Composites Loaded

with Nano/Micro Structured Clays (Dellite, D – Montmorillonite) and Synthetic Lamellar

Perkalite (LDH).

Figure 4.24. Torque Behaviour vs. Time Recorded for PHBV Samples Loaded with Joncryl

at Different Concentrations (0.2, 2 and 5% by Weight).

Figure 4.25. Torque Behaviour vs. Time Recorded for the Hybrid PHBV Based Composites

Loaded with Lignin (LN) and Dellite (D).


- 122 -

Figure 4.26. Torque Behaviour vs. Time Recorded for the Hybrid PHBV Based Composites

Loaded with Lignin (LN), Dellite (D) and Joncryl (J).

In general, from the Figures 4.21 to 4.26, it has been observed that all the additives blended

with the PHBV in binary and ternary system decreases substantially the starting torque value

(during feeding) and the final one (trial end) compared to neat PHBV processed in the

Brabender (see Table 4.8). This result confirms that the choice of additives and organic

and/or inorganic fillers was appropriated for PHBV and they don‘t constitute components

that can give adverse reactions such as degradation and cross-linking in the melt.

From the Figure 4.22 it is possible to observe that the hazelnut shell powder (lignin) with

particle dimensions below 39 micron and concentration 5% is the only additive that shows

similar torque behaviour as PHBV starting raw material.

Organic PHBV based composites loaded with lignin (10 and 20%) and the blend containing

Joncryl at 5% and the inorganic composite loaded with lamellar double hydroxide (LDH) (at

3% by wt.) show the major torque stability (see Figure 4.21-4.24). The minor final torque

value is observed for the sample PHBV + LDH3 (Table 4.8).

Moreover, the hazelnut shell powder was used without any treatment of purification and

initial fractionation. So some fractions can contain natural antioxidant agents that can

stabilize PHBV polymer matrix during its processing (see Figures 4.21 and 4.22), thus

protecting it from its thermal degradation via a crotonization reaction, responsabile of a

molecular weight decrease for the starting biopolymer. The lignin addition is useful also for

(1)Rreducing the retail price for various composite formulations, (2) Reinforcing the PHBV

mechanical properties.

Figure 4.24 shows the effect of the Joncryl dispersion on the torque stability and process

ability of pristine PHBV polymer. The Joncryl dispersion in the polymeric matrix shows a

small stabilization effect on the torque during the entire resident time. Considering that the

Joncryl is an additive useful to rebuild the polymer chains in reground condensation

polymers and to restore the melt strength of the polymer into a level sufficient for stable

processing, and for good mechanical strength of the polymer, can be expected an increment

of the torque during the PHBV processing as has been observed during the PLA processing

(see Figures 4.31 and 4.32). Contrary, the variation of PHBV torque was less than expected

and two possible explanations have been put forward:

1) The high molecular weight of PHBV polymer makes unfavourable the chain

extension reaction between the reactive terminal groups from PHBV with the epoxy

groups from Joncryl additive due to a dissolution effect of the end chains reactive

groups inside the melted polymer bulk.


- 123 -

2) When the processing temperature is near to the depolymerization temperature of the

polymer, many reactions can have been occurred: the reaction between the Joncryl

epoxidic groups with the reactive site of crotonic acid (obtained from the PHBV

thermal degradation) becomes a competitive reaction with the terminal groups of

the PHBV and it doesn‘t give a relevant effect on the torque evaluation caused by

the small dimensions of the crotonic acid molecules.

However, the Joncryl effect on PHAS results in the combination of two or more

contemporary factors, which become relevant considering the distribution of polymer

molecular weight, the starting molecular weight and the processing conditions.

Deep study must be conducted in order to elucidate the real mechanism of Joncryl on the

PHBV chains during the melt processing and the factor that affects it.

Table 4.8. Final Torque and Stock Temperature Values Recorded for PHBV Based Blends

and Composites.

Sample Code Final Torque

(N*m)

Stocka)

Temperature

(°C)

PHBV 10.1 174

PHBVJ0.2 6.2 175

PHBVJ2 5.3 174

PHBVJ5 5.0 174

PHBVLN5 11.2 176

PHBVLN10 8.2 175

PHBVLN20 9.5 175

PHBVLN10J5 9.5 173

PHBVLN20J5 8.5 174

PHBVLDH3 3.2 173

PHBVD3 5.7 174

PHBVD6 4.4 174

PHBVD3J2 4.9 174

PHBVLN10D3 5.4 174

PHBVLN20D3 6.9 175

PHBVLN10D3J2 5.8 174

PHBVLN20D3J2 6.5 174

PHBVLNF10 0.7 170

a) Stock temperature = set up temperature + 20°C

Functionalized lignin was prepared (see chapter 2) with the aim to improve the compatibility

between the lignin flour, collected from grinding hazelnut shells, and the hydrophobic PHBV

. By using functionalized lignin (LNF), it is possible to observe from Figure 4.27 a dramatic

drop in the torque from the beginning until the end of the processing experiment. This

behaviour has been attributed to a loss on the PHBV molecular weight, suggesting that this

modified organic filler is not useful for maintaining the molecular weight integrity during the

processing operation. This could be correlated with some degradation reactions between the

hazelnut shells components and the modified lignin. This lignin results an unstable

component for the presence of esters groups: the obtained composite resulted very fragile.


- 124 -

Figure 4.27. Torque Behaviour vs. Time Recorded for PHBV Based Composites loaded with

Lignin (LN) and functionalized Lignin at 10% by wt.

Figure 4.28 shows the dispersion effect promoted by Joncryl on the torque stability and the

PHBV processing. The additive dispersion in the PHBV powder, produces a small stabilizing

effect for the torque during the whole residence time (7 minutes). Joncryl is an additive able

to regenerate the polymeric chain lengths in condensation polymers, giving to the polymer a

good strength in the melt. So we can use stable processing conditions best suited for the

attainment of the required mechanical properties for the final composite material .

Figure 4.28. Torque Behaviour vs. Time Recorded for PHBV/Joncryl at 5%- Assessment of

the Joncryl Dispersion.

In terms of processing in the melt, the most suitable concentration in %-wt for each additive

is reported by following (Table 4.9).

Table 4.9. Better Components Percentage as Ranked on the Torque Response

Lignin (LN) 10

Perkalite (LDH) 3

Dellite (D) 6

Chain extender (Joncryl- J) 0.2 -2


- 125 -

Figures 4.29, 4.30 and 4.31 shows the PLA processing characteristic related with the

influence of Joncryl and lignin additives. Figure 4.32 shows the torque behaviour for the

composites varying the amount of lignin by keeping constant the amount of Joncryl. Figure

4.33 shows a comparison for the torque behaviour for the most representative PHBV based

composites vs. PLA based composites.

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6 7 8

Time (min.)

To

rqu

e (

N*m

)

PLA

PLA + J0.2

PLA + J2

PLA + J5

Figure 4.29. Torque Behaviour vs. Time Recorded for PLA Samples Loaded with Joncryl at

Different Concentrations (0.2, 2 and 5% by Weight).

10

11

12

13

14

15

16

17

18

19

20

0 1 2 3 4 5

Time (min.)

To

rqu

e (

N*m

)

PLA

PLA + J0.2

PLA + J2

PLA + J5

Figure 4.30. Enlargement of Torque Behaviour vs. Time Recorded for PLA Samples Loaded

with Joncryl at Different Concentrations (0.2, 2 and 5% by Weight).

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6 7 8

Time (min.)

To

rqu

e (

N*m

)

PLA

PLA + LN10 up 39 microns

PLA + LN10 under 39 microns

PLA + LN20 up 39 microns

PLA + LN20 under 39 microns

Figure 4.31. Torque Behaviour vs. Time Recorded for PLA Composites Loaded with Lignin

at Different Concentrations and Size (10 and 20% by Weight).


- 126 -

Figures 4.32 and 4.33, show the effect of the Joncryl dispersion and concentration on the

torque stability and processability of PLA pristine polymer. The Joncryl dispersion in the

polymeric matrix shows a small stabilization effect on the torque during the entire resident

time. In particular, a notorious increment in early torque values (Figure 4.33) constitutes an

experimental evidence for the reaction between Joncryl and PHBV when the amount of

Joncryl increases from 2 until 5%.

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6 7 8

Time (min.)

To

rqu

e (

N*m

)

PLA

PLA + LN10 + J5

PLA +LN20 + J5

Figure 4.32. Torque Behaviour vs. Time Recorded for PLA Samples Loaded with Joncryl at

5% by Weight and Different Amount of Lignin (LN).

Figure 4.32 shows the torque behaviour for PLA based composites blended with different

amount and size of lignin. By varying the amount and the size of the organic filler was not

possible observe significant changes in the PLA torque behaviour and the results reported in

Table 4.10 confirm this tendency.

Figure 4.33 shows the torque behaviour for PLA based composites having different amount

of lignin and constant amount of Joncryl. The sample containing 20% of LN and 5% of

Joncryl presents the higher torque stability during the processing, suggesting that this

composite is a suitable candidate for further formulations.

Table 4.10. Final Torque and Stock Temperature Values Recorded for PLA Based Blends &

Composites.

Sample Code Final Torque

(N*m)

Stocka)

Temperature

(°C)

PLA 8.3 186

PLA + J0.2 11.0 189

PLA + J2 13.0 251

PLA + J5 12.0 191

PLA + LN10 39 µm 8.0 187

PLA + LN20 39 µm 7.0 186

PLA + LN10 < 39 µm 7.1 187

PLA + LN20 < 39 µm 9.5 188

PLA + LN10 + J5* 13.4 191

PLA + LN20 + J5* 15.6 193

a) Stock temperature = set up temperature + 20°C

* LN < 39 µm

The effect of the Joncryl and the organic filler on the rheological characteristic of PHA‘s

composites are more evident in PLA matrix than PHBV matrix (see Figure 4.34).


- 127 -

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6 7 8

Time (min.)

To

rqu

e (

N*m

)

PHB

PLA

PHB + LN10 + J5

PLA + LN10 + J5

PHB + LN20 + J5

PLA + LN20 + J5

Figure 4.34. Torque Behaviour vs. Time Recorded for PHBV Samples loaded with Joncryl at

Different Concentrations (0.2, 2 and 5% in Weight).

4.5. PHBV Based Blends and Composites Structural Characteristic by Infrared

Spectroscopy (IR)

Figures 4.35-4.37 show the IR spectra recorded for pure PHBV sample and its blends and

composites loaded with the organic filler (lignin), the chain extender (Joncryl) and an

organophilic clay such as montmorillonite named Dellite.

Figure 4.35. IR Spectra Recorded for Pure PHBV Sample and PHBV/Joncryl Blends.


- 128 -

(a) (b)

Figure 4.36. IR Spectra Recorded for Pure PHBV Sample, PHBV/Lignin Blends and

PHBV/Lignin/Joncryl Composites.

Figure 4.37. IR Spectra Recorded for Pure PHBV Sample and PHBV/Clay (Dellite) Blends.

Figure 4.35 shows the effect of Joncryl concentration on PHBV matrix. The position and the

shape of the IR bands remain unalterate, whereas the intensity of same bands are different in

the analyzed spectra when we consider the neat polymer or its blends and composites

containing an organic filler, a synthetic additive or an organophilic clay.

The addition of the organic filler (lignin), the chain extender (Joncryl) and the organophilic

clay (Dellite) do not create any relevant shift for the C-O, C=O and O-H stretching bands for

all PHBV based blends and composites compared to neat PHBV, except for the blend

PHBV/J0.2 and the composites PHBV/LN20, PHBV/LN10/J5 and PHBV/LN20/J5.

In their IR traces, the IR band correlated to the C=O stretching, falls down at lower

wavenumbers than the value recorded for the same band of neat PHBV: this effect can be

attributed to the resonance effect caused by the epoxy ring in the case of the blend

PHBV/J0.2 and by the aromatic rings in the the case of the hybrid composites (PHBV/LN/J).

Figure 4.38 reports the IR spectra recorded for PLA pure sample and its blends and

composites loaded with the organic filler (lignin) and the chain extender (Joncryl).


- 129 -

(a) (b)

Figure 4.38. IR Spectra recorded for Pure PLA Sample, PLA/Joncryl Blend (a) and

PLA/Lignin/Joncryl Composite (b).

The addition of lignin alone or in combination with Joncryl decreases the intensity of the

band correlated to -CH2 stretching band and the shape of the band correlated to -OH

stretching one.

These additives do not modify in a consistent manner the remained part of the spectra.

The only difference, that we can detect, regards the IR spectra recorded for PLA pure sample

and the blend PLA/J containing the additive in a percentage of 5 by weight. The epoxy rings

contained in the additive chains shift to upper value the band correlated to the -C-O

stretching for this selected blend.

4.6. Biodegradation Experiments

The assessment of the extent of biodegradation under different conditions (soil, compost and

water river) for the bio-based polymer composites, constitutes another step to better know

the ultimate environmental fate of these materials and the effect of each component,

comprised in the composite, on the propensity to biodegrade of the material. The requirement

of two steps, abiotic and biotic, in the degradation mechanism of biodegradable plastic

materials has recently led to the preparation and approval of ASTM D5988-03 ―Standard

Test Method for Determining Aerobic Biodegradation in Soil of Plastic Materials or

Residual Plastic Materials After Composting‖. This standard provides a framework to assess

and compare the determining aerobic biodegradation in soil of polymers or residual plastic

materials after composting as well as the degree of biodegradation and ecological impacts in

defined environments after abiotic degradation.

All the experiments have been set up according to standardized procedures relevant to the

evaluation of the biodegradability of plastic items in soil (ISO 17556), compost (ISO 14855)

and aqueous medium (OECD 301B).

The biodegradation in mature compost have been carried out up to completion (6 months of

incubation), whereas the biodegradation tests in soil and water media are still ongoing even

though in a advanced stage with respect to the incubation time frame suggested by the

adopted standardized test procedures.

The results so far collected are thus allowing to formulate conclusive remarks relevant to the

effects on the biodegradation propensity of the analyzed materials as promoted by the

additives and fillers used in the formulation of PHBV based blends and composites. In

particular ligno-cellulosic fillers exhibited a positive effect on the rate and extent of

biodegradation particularly in solid media (soil and mature compost) tests, whereas the

polymeric chain extender additive (Joncryl ADR-4368C) was promoting a general

depressive effect, whose extent was correlated to the amount used in the material formulation.


- 130 -

4.6.1 PHBV Based Blends and Composites in Soil Medium

In the soil burial respirometric test, the biodegradation kinetics of several PHBV based

composites were compared with those of a pure PHBV sample, with the aim to investigate

the influence of additive and fillers utilized in the composites preparation. In particular, it

was possible to analyze the effect of several components such as the organic filler (lingo-

cellulosic material from hazelnut shells), the inorganic filler (a modified montmorillonite

named Dellite 72T), and special additives such as the chain extender Joncryl ADR-4368 on

the kinetic of the biodegradation process. The analyzed samples, their organic carbon content

and the relevant theoretical CO2 amounts are collected in Table 4.11.

Table 4.11. Organic Carbon Content and Theoretical CO2 for Relevant PHB Based Blends

and Composites.

N Test Sample Code Weight Sample 1

(mg)

Weight Sample 2

(mg)

C org

(%)

ThCO2

(mg)

1 PHBV 382.4 380.1 57.5 803.1

2 PHBV-LN20-D3-J2 380.7 380.3 55.6 775.0

3 PHBV-LN20 381.5 380.4 55.1 768.9

4 PHBV-LN20-J5 380.8 380.0 53.9 751.8

5 PHBV-LN20-D3 380.1 380.5 55.3 770.4

6 PHBV-D3-J2 380.8 381.1 56.1 782.9

7 PHBV-LN10-D3-J2 380.3 382.6 54.5 762.3

8 PHBV-LN10 381.7 380.0 55.8 778.5

9 PHBV-LNF10 384.1 381.6 56.0 786.1

10 PHBV-D3 380.8 384.7 56.6 793.6

11 PHBV-J5 380.3 382.4 59.1 825.7

12 PHBV-LN10-J5 381.3 382.7 56.8 794.9

13 PHBV-LN10-D3 381.2 384.2 55.9 784.4

14 Cellulose 382.2 381.6 44.4 621.7

In Figure 4.40 (a-c), the biodegradation profile of the analyzed materials and reference

compound (cellulose) collected within 12 months of incubation, are reported. The collected

results suggested that the addition of the ligno-cellulosic filler improved the rate and extent

of biodegradation, as recorded in the case of PHBVLN20 sample, whose extent of

mineralization approached a 90% level within the incubation time, whereas the pure PHBV

sample did not exceed 70% biodegradation in the same time frame (Figure 4.40a).

Comparable effects were also recorded in the case of composites containing the combination

of organic and inorganic (Dellite 72T) fillers (Figure 4.40c). Moreover, the biodegradation

trends were similar in the case of the composites having 10% and 20% by weight of hazelnut

shells (lignin), thus suggesting that the biodegradation trend is not affected by the selected

concentrations. On the other hand, the presence of significant amount of the chain extender

additive reduced the rate and the extent of the mineralization process independent of the

presence of organic and inorganic fillers, thus exhibiting a stronger effect as a function of the

concentration utilized in the composites processing (Figures 4.40a, 4.40b and 4.40c).


- 131 -

(a) (b)

(c) (d)

Figure 4.40. Mineralization Trend for PHBV/Lignin Composites (a); PHBV/Lignin/Joncryl

Blends (b); PHBV/Inorganic/Organic Filler Composites (c), PHBV/Inorganic/Organic

Filler/Joncryl Composites (d).

Biodegradation takes place through the action of enzymes and/or chemical deterioration

associated with living organisms. This event occurs in two steps, that are the fragmentation

of the polymer into lower molecular weight species by means of either abiotic reactions, such

as oxidation, photo-degradation, hydrolysis, or biotic reactions, such as degradations

promoted by microorganisms. This is followed by bio-assimilation of the polymer fragments

by microorganisms and their mineralisation.

Biodegradability depends not only on the origin of the polymer but also on its chemical

structure and the environmental degrading conditions (Vroman 2009).

The most important factors related to the PHAs characteristics that influence their

degradation are (a) microtacticity, since only monomers in configuration (R) are hydrolyzed

by depolymerases, (b) crystallinity, since the degradation decreases with higher crystalline,

(c) molecular weight, because the polymer of low molecular weight are generally degraded

more rapidly than those with high molecular weight, (d) monomeric composition of PHAs

(Lutz and Falcao 2011).

Also the composition and the compatibilization between the components inside the

formulation play an important role in the biodegradation process for PHBV based

composites loaded with organic filler. In our case, the lignin addition can make more

favourable the hydrolytic degradation of PHBV polymer matrix, even if lignin was found to

exert a protective function against PHBV biodegradation (Mousavioun et al. 2012).

The mineralization trend for PHBV containing melts strength enhancer like Joncryl ADR-

4368 alone or together with lignin showed an inhibitory effect. It can be hypothesized that

the additive promoted a scavenging activity connected to the epoxydic groups responsible of

a possible antibacterial and antifungal activity, thus making very slow the biodegradation

process for PHBV/Joncryl based composites. On the other hand it can be also hypothesized

that the detrimental effect on the biodegradation process could be determined by the


- 132 -

structural modification (e.g. cross-linking) promoted by the Joncryl addition. A slight, but

significant improvement in the biodegradation process in soil was also observed when PHBV

was processed in the presence of an inorganic component such as Dellite 72T.

Dellite is a modified montmorillonite with quaternary ammonium salts, it can be thereof

hypothesized tha the nitrogen release from the PHBV/Dellite composites, may act a fertilizer

action towards the microorganisms population in the soil (Burkart et al. 2005), making more

favorable the mineralization process.

4.6.2. PHAs Based Blends and Composites Biodegradation in Mature Compost

In the mature compost respirometric tests, the biodegradation kinetics of several PHBV

based composites were compared with the same ones submitted to the biodegradation

experiments in soil, with the aim to investigate the influence of additives and fillers utilized

in the composites preparation on the kinetic of the biodegradation process, as well as to

evaluate the influence of the composting environment with respect to the soil.

The values of total organic carbon content (TOC) and relevant theoretical evolution of

carbon dioxide (ThCO2), necessary to obtain the mineralization percent versus time for the

investigated samples, are collected in Table 4.12.

Table 4.12. Organic Carbon Content (TOC) and Theoretical CO2 (ThCO2) for PHBV Based

Blends & Composites.

N Test Sample Code Weight

Sample 1

(mg)

Weight

Sample 2

(mg)

C org

(%)

ThCO2

(mg)

1 PHBV 146.4 134.1 57.5 295.7

2 PHBV-LN20 148.3 142.5 55.1 293.8

3 PHBV-LN20-J5 146.1 148.3 53.9 290.9

4 PHBV-J5 145.0 149.2 59.1 318.8

5 PHBV-J0.2 149.2 167.3 57.7 334.8

6 Cellulose 144.7 153.7 43.9 240.2

Figure 4.41 shows the mineralization trend for PHBV based composites in compost medium.

(a) (b)

Figure 4.41. Mineralization Trend for PHBV/Joncryl Blends (a) and PHBV/Lignin

Composites (b).

The detrimental effect upon Joncryl addition on PHBV polymer matrix on the mineralization

process of the analyzed materials recorded during the soil burial respirometric test was also


- 133 -

observed during the mature compost test (Figure 4.41a). The effect was dramatic once the

concentration of the additive reached 5% by weight (Figure 4.41a). On the other hand, the

presence of ligno-cellulosic filler was found to mitigate the negative effect of the chain

extender additive. Under the adopted incubation condition, the PHBV composite containing

both Joncryl and the organic filler underwent to an extensive mineralization process even

though exhibiting a multi step biodegradation profile (Figure 4.41b).

4.6.3. PLA Based Blends and Composites Biodegradation in Mature Compost

The effect of Joncryl concentration is showed in Figure 4.42a whereas the effect of the

organic filler addition on the biodegradation propensity for PLA polymer matrix is depicted

in Figure 4.42b.

The values of total organic carbon content (TOC) and relevant theoretical evolution of

carbon dioxide (ThCO2), necessary to obtain the mineralization percent versus time for the


Table 4.13. Organic Carbon Content (TOC) and Theoretical CO2 (ThCO2) for PLA Based

Blends & Composites.

N Test Sample Code Weight

Sample 1

(mg)

Weight

Sample 2

(mg)

C org

(%)

ThCO2

(mg)

1 PLA 165.0 154.0 49.7 290.7

2 PLA-LN20 156.0 147.0 50.4 280.0

3 PLA-LN20-J5 157.0 142.0 50.8 278.5

4 PLA-J5 148.0 145.0 51.2 275.0

5 PLA-J0.2 156.0 147.0 52.2 300.5

6 Cellulose 144.7 153.7 43.9 240.2

(a) (b)

Figure 4.42. Mineralization Trend for PLA/Joncryl Blends (a) and PLA/Lignin Composites

(b).

The biodegradation process for PLA based blend containing Joncryl in low concentration

(0.2%) presents a light inhibition respect to the pure PLA sample, as observed from the

traces that remain below the PLA trace (Fig. 4.42a). The same trend can be observed for the

trace of PLALN20J5 composite, where the organic filler try to mitigate the inhibition created


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by Joncryl. The additive concentration increasing until 5% has a dramatic effect on the

biodegration propensity of PLA matrix, in the same manner observed for PHBV

biodegradation pathway.

The chain extender Joncryl posses an antibacterial activity able to decrease the number of

microorganisms, that can degrade the polymer (Mousavioun et al. 2012).

Figure 4.43 reports the hydrolitic degradation mechanism for PLA, in which the molecoles of

water adsorbed by the polymer are able to break down the ester linkages in the polymer

chains. This process can be auto-catalyzed by terminal carboxilic groups and it follows a first

order kinetic, that is influenced by the starting cristallinity, the sample widht and shape

(Siracusa et al. 2008).

Figure 4.43. Hydrolytic Degradation Mechanism for PLA.

The presence of lactic acid and low molecular weight products show an important role in

promoting the starting of a biodegradation process for a film, because of fungi and bacteria

assimilate the inizial lactic acid, colonizing the film superficial area (Hakkarainem 2000).

4.6.4 Qualitative Compost Experiment

In Figures 4.42 and 4.43 the pictures of the most representatives PHBV based composites

samples analyzed for the determination of the degree disintegration within 45 days

incubation, are reported. The test was carried out in accordance with the ISO 14855

normative. More recent norms such as EN-13432-02, ASTM6400-04 and ISO 17088, are

suggesting to increase the incubation time up to twelve weeks (84 days).

After 37 days of incubation, an appreciable growth of filamentous microorganisms (i.e. fungi

and actinomycetes) was observed on the PHBVLN10 and PHBVLNF10 specimens surface

(Figures 4.44 and 4.45). The observed microbial growth was accompanied by a marked

discoloration of the samples specimens (Figure 4.44), whereas no significant trace of

microbial deterioration was observed in the case of PHBVD3 and PHBVJ5 specimens,

containing the inorganic filler and the chain extender, respectively. These observations were

thus suggesting that the samples, containing 10 % by weight of ligno-cellulosic filler and

esterified lignin, are more susceptible to the microbial attack.

In Table 4.14, the gravimetric analysis of the tested samples is reported. The collected data

confirmed the observation obtained by the visual inspection of the analyzed specimen. In

accordance significant specimen weight losses ranging between 2.5-6.0 of the original


- 135 -

weight were recorded after 45 days of incubation only in the case of samples processed with

the organic fillers.

The collected observation are suggesting the microbial attack of PHBV based composites

proceeds via surface erosion in accordance with the results obtained by Yun-Xuan Weng et

al (2010) that analyzed the biodegradation behavior of PHBV at pilot scale composting

conditions.

The qualitative results suggested that the presence of lignin can modify the characteristic of

the PHBV based composites leading to a surface more compatible with the surrounding

media and the microbial adhesion thereof.

Table 4.14. Weight Variations During the Biodegradation in Compost for PHBV Most

Representative Composites

Sample Initial Weight at

t=0 (mg)

Final Weight at

t=45 (mg)

Variation (%)

PHBV + LN10% 320,1 311,2 2,8

PHBV + LNF10% 306,4 287,7 6,1

PHBV + D3% 329,2 325,5 1,1

PHBV + J5% 356,2 356,3 0

Figure 4.44. Evolution of the Disintegration Within 45 Days (Setup 1)


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Figure 4.45. Evolution of the Disintegration Within 45 Days (Setup 2)

a) Organic filler b) Inorganic filler

Figure 4.46. Variation of the Colors Sample for PHBV Composites.

4 .6.5. River Water Respirometric Test

The biodegradation experiment in aqueous medium was performed on selected PHBV based

blends and composites in order to investigate the influence of the organic filler (ligno-

cellulosic material from hazelnut shells) and special additives on the kinetic of the

biodegradation process. The propensity to biodegradation in aqueous medium of selected

PHBV based samples was compared with those of reference materials (cellulose and sodium

acetate, CH3COONa), as well as with a low density polyethylene sample (Riblene FL30) and

commercial PLA.

The values of total organic carbon content (TOC) and of the theoretical evolution of carbon

dioxide (ThCO2), necessary to obtain the mineralization percent versus time for the


PHBV-LNF10 PHBV-LN10 PHBV-D3 PHBV-J5


- 137 -

Table 4.15. Organic Carbon Content (TOC) and Theoretical CO2 (ThCO2) for PHB Based

Composites and Formulations, References.

Test Sample Code Organic Carbon (TOC)

(%)

ThCO2

(mg)

PHBV 57.5 110.3

PHBV-J0.2 57.7 101.9

PHBV-J5 59.1 108.1

PHBV-LN20-J5 53.9 99.6

PLA 49.7 88.2

Cellulose 43.9 81.3

Riblene FL30 85.6 154.1

CH3COONa 29.2 54.6

In Figure 4.47a-c the biodegradation profiles recorded within 90 days of incubation in

aqueous medium of PHBV based composites and reference materials are reported.

(a) (b)

(c)

Figure 4.47. Mineralization Trend for PHBV/Joncryl Blends (a), PHBV/Lignin/Joncryl

Composite (b), References (c).

Under the adopted incubation conditions, the detrimental effect of relatively high

concentration (5% by weight) of the chain extender Joncryl was confirmed (Figure 4.47a and

4.47b). It was also observed that lower concentration (0.2%) of the additive did not affect

significantly the rate and extent of PHBV biodegradation (Figure 4.47a).

The organic filler did not try to mitigate the inhibition effect created by Joncryl at high

concentration (5%) on PHBV biodegradation propensity (Figure 4.47b).

Finally it is interesting to note the negligible, if any, biodegradation of the ―biodegradable‖

polyester PLA, whose propensity to microbial attack in aqueous medium at room


- 138 -

temperature resulted strictly comparable with tha exhibited by the not-biodegradable LDPE

sample (Figure 4.47c).

4.7. Biodegradation of Fossil Fuel Based Polymers (ECOFLEX, PE and PS) in Soil-

Plant Systems (Avena sativa –Raphanus sativus)

In the present investigation the potential eco-toxicological effects in soil-plant systems of

polymeric materials represented by thermally degraded Polyethylene (PE) and Polystyrene

(PS) containing proprerly pro-degradant additives were studied; the effects exerted by PE

and PS polymers were compared with those of biodegradable materials such as Ecoflex and

Cellulose, used as positive controls.

The definition of the environmental parameters to be considered when planning a soil

biodegradation test is the first dilemma encountered by researchers. This problem is less

critical when defining biodegradability under composting conditions, because the variability

of the composting environment is low. The composting environment is a rather

homogeneous ecological niche and can be considered as a consistent micro-cosmos. On the

other hand, the environmental factors in soil can be very different from one location to

another and consequently the rate of degradation can be different. Therefore, when studying

biodegradation in soil, characterization of the environmental factors can be important to

relate the biodegradation behavior to a specific soil.

4.7.1. Potential Eco-toxicological Effects of Polymer Materials

The values of seed germination and plant growth were reported in Table 4.16.

Table 4.16. Plant germination and growth measured after 10 days from seedling (45 day

from the beginning of the experimentation)

Sample

Seed

germination

(%)

Average Plant

Length

(mm)

Avena 67 106

Cellulose+Avena 73 103

PE+Avena 74 104

PS+Avena 85 132

Ecoflex+Avena 77 115

Radish 92 53.9

Cellulose+Radish 82 39.3

PE+Radish 70 57.1

PS+Radish 80 49.0

Ecoflex+Radish 65 45.4

The results suggested that the germination of the Avena sativa seeds is not negatively

affected by the application of biomaterials, while for Raphanus sativus there is evidence of a

light inhibition played by all polymers. As regards Avena sativa system, the germination

percentage in degraded polystyrene (PS) treated soil sample was higher than in the other

treatments; moreover, the values of the polymer treatments were always higher than the


- 139 -

untreated soil sample (Avena sativa). On the contrary, the soil Raphanus sativus control

system showed the highest value, with the other treatments being in the order:

Cellulose=PS>PE>Ecoflex. The results of average plant lengths reflected the germination

percentage, confirming the different sensitivity of the two selected plant species.

In fact, based on the GI % parameter (Fig. 4.48), a negative effect of polymer application on

soil plant germination and growth is only evident in the presence of Raphanus sativus.

Therefore, in order to give assessment of eco-toxicological effect in a short period, Raphanus

sativus should be considered as a more appropriate indicator.

Fig. 4.48. Plant Germination and Growth Index (GI%).

4.7.2. Soil biochemical parameters

Two categories of microbial enzymes are involved in the degradation of plastic polymers,

namely extracellular and intracellular enzymes. Exoenzymes from microorganisms first

breakdown the complex polymers giving short chains that are small enough to permeate

through the cell walls to be utilized as carbon and energy sources. The process is called

degradation. When the end products are carbon dioxide, water or methane, the process is

called mineralization.

In the degradation of polymers, enzymes function as catalysts for a specific reaction or a

series of reactions, such as oxidation, reduction, hydrolysis, esterification, synthesis and

molecular inter-conversions. Hydrophobic polymers with no hydrolyzable bonds such as PE

and PS are expected to be the most stable (Zeng et al., 2005).

Several soil factors including the availability of water, temperature, oxygen availability,

minerals, pH, redox potential, carbon and energy source influence the growth and activity of

microorganisms (Sand 2003). All these factors, joined together in different combinations,

create different environments and strongly affect the soil ecology. As a consequence the

microbiology and the biodegradation activity can change from soil to soil and from season to

season.

Soil enzyme activities have been considered as useful markers of the impact of pollutants on

the metabolic activity of soil (Ceccanti et al. 2006; Doni et al., 2012). They are the catalysts

of important metabolic functions, including the decomposition and the detoxification of

contaminants; they can rapidly change in response to changes in soil caused by both natural

and anthropogenic factors and they are fairly easy to measure (Nannipieri et al. 2002). As a

result of these advantages, it has been suggested that soil enzyme activities are useful as

early and sensitive indicators of soil alteration in both natural ecosystems and ecosystems


- 140 -

altered by anthropogenic activities, and, in addition, they are well suited to measure the

impact of pollution on soil quality.

Phosphatases are a large group of intra- and extra-cellular enzymes in soil related to the

phosphorus cycle. They are essential for plant nutrition, since catalyze the hydrolysis of

phosphoric esters releasing inorganic phosphate. Most of phosphorus in soil is in organic

form, and this results from the decomposition of nucleotides, proteins, phospholipids or other

soluble organic compounds. Plants absorb this element in inorganic form through their root

system. The mineralization of organic compounds containing phosphorus is performed by

phosphatases in the nutrient medium or by the root surface. These enzymes play a

particularly important role in soil since that catalyze reactions from different types of

substrate despite having a low specificity (Alef and Nannipieri, 1995).

In the present experimentation (Table 4.17), phosphatase activity generally increased in the

presence of both plant species, Avena sativa and Raphanus sutivus, from T0 (planting time)

to T1 (ten days after planting), suggesting a stimulation of microbial metabolism at root level.

The Ecoflex treatment is the only one that showed a decrease in this enzyme activity. The

highest phosphatase activity was shown in the cellulose treatment with Raphanus sativus

plant at T1.

Protease activity showed very little variation; in general, higher values were observed in the

polymer treatments with respect to the untreated soil. Proteases are a group of hydrolytic

enzymes associated with the nitrogen cycle; they are involved in the first reaction of the

metabolic sequence responsible for the transformation of protein nitrogen into ammonia

(mineralization). As previously reported for phosphatase activity, also protease showed the

lower values in the Ecoflex treatments.

Table 4.17. Enzymes Activity from T0 (Planting Time) to T1 (Ten Days After Planting) for

Some Polymers and Plants Treatments.

Protease

(mgNH3/kg*h)

Phosphatase

(mgPNP/kg*h)

DH-ase

(mgINTF/kg*h)

T0 T1 T0 T1 T0 T1

Control 17.5 16.2 254 226 3.59 3.52

Avena 20.1 20.2 212 234 3.61 2.83

Cellulose+Avena 23.0 24.3 227 240 4.27 4.63

PE+Avena 19.0 20.7 207 257 3.27 5.48

PS+Avena 20.7 23.6 213 281 3.27 3.79

Ecoflex+Avena 16.4 14.9 220 191 4.27 4.54

Radish 18.2 16.3 202 231 3.42 3.20

Cellulose+Radish 22.1 20.1 243 452 4.57 4.70

PE+Radish 22.4 20.1 239 313 2.01 4.63

PS+Radish 20.7 20.8 181 276 2.75 3.46

Ecoflex+Radish 18.7 18.4 238 181 3.23 3.41

T0, soil samples after 35 days from the beginning of experimentation (50% of water

retention capacity; 25 °C of temperature); T1, soil samples after 45 days from the beginning

of experimentation.


- 141 -

Dehydrogenases are principally intracellular enzymes and belong to the group of

oxidoreductases; they catalyze the oxidation of organic compounds with the removal of two

hydrogen atoms that are transferred to the molecule of NAD+ or NADP+ (Nannipieri and

Bollag 1991). Dehydrogenase activity is therefore a good indicator of the redox

microbiological system and can be considered a good measure of microbial oxidative activity

in soil (Laudicina, et al. 2012; Masciandaro, et al. 2000).

In the present experimentation, dehydrogenase activity increased over time in all the

treatments, while decreased or remain stable in the unplanted (control) and in the untreated

planted soil (Avena sativa and Raphanus sativus) (Table 4.17 and Figure 4.49), thus

indicating an effect of the polymer addition on microbial metabolism stimulation. This

microbial activity stimulation can be responsible for the hydrolytic enzyme activity increase,

especially phosphatase.

Fig. 4.49. Dehydrogenase Activity of Soil Samples.

The metabolic index, calculated as the ratio between dehydrogenase activity and water

soluble carbon (DH-ase/WSC) represents a parameter related to metabolic activity and

availability of easily degradable organic carbon, which represents also a source of nutrients

(Masciandaro et al., 2004).

This parameter seems to be stimulated at T1 in the presence of both plant species. This trend

indicates a general improvement of soil metabolism being both plants capable to remove the

inhibitory effect of polymer in soil.

Fig. 4.50. Metabolic Index of Soil Samples.


- 142 -

Even though with these laboratory tests it is not possible to prove biodegradation in terms of

metabolization by microorganisms, the short and reproducible test conditions make them

especially useful for a preliminary assessment and comparison of polymer biodegradability

in soil.

4.7.3. Chemical parameters of soil samples treated with different polymers

Soil chemical parameters measured during the experimentation were shown in Table 4.18.

The pH values did not show evident differences in the biopolymer treated soil samples with

respect to the control. However, a general increase in electrical conductivity was observed at

T1 sampling time in the presence of Raphanus sativus, in particular in the treatment with

cellulose and polyethylene. The higher electrical conductivity in these soil samples is

probably due to the higher content of nitrate, which reached the highest value in the cellulose

treatment. This suggests the establishment of the better aeration conditions for

microorganisms in carrying out their activity. In agreement with this result, the Raphanus

sativus planted soil in the presence of cellulose showed, at T1 sampling time, the high

dehydrogenase activity.

These results suggest the positive effect of legume plant Raphanus sativus towards microbial

activity and N cycle. Both plant species improve the availability of the nutrients, usually

expressed by the ratio nitrate/ammonia ions. The WSC showed a different trend; the lower

values were, in fact, generally observed in correspondence of higher values of nitrate. In

view of this, a negative correlation nitrate/WSC has been found in presence of Raphanus

sativus (Table 4.18; r=-0.639).

These results clearly indicated the best capability of Raphanus sativus in reducing water

soluble carbon (WSC) during polymer degradation in soil, which prevent loss of carbon and

a potential surface and ground water pollution.


- 143 -

0, soil samples after 35 days from the beginning of experimentation (50% of water retention capacity; 25 °C of temperature); T1, soil samples after 45

days from the beginning of experimentation.

Tab. 4.18. Chemical Parameters of the Soil in the Different Treatments

pH Electrical

Conductivity (µS/cm)

Water soluble

carbon (WSC)

(mgC/kg)

NO3-/NH4

+

Nitrate

(mgNO3-/Kg)

Ammonia

(mgNH3/Kg)

T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1

Control 7.1 7.2 106.3 113 466 455 18.26 14.6 225.12 175.09 12.33 12.0

Avena 7.04 7.31 109.4 95.7 391 391 11.18 12.62 178.64 140.26 15.98 11.12

Cellulose+Aven

a 7.02 7.22 127.3 118.9 445 649 14.22 10.79 229.97 158.44 16.18 14.69

PE+Avena 7.17 7.21 123 118.3 670 455 34.75 28.21 263.23 296.49 7.58 10.51

PS+Avena 7.07 7.18 128.1 125.7 466 455 15.3 22.89 320.23 314.69 20.93 13.75

Ecoflex+Avena 7.02 7.1 116.9 51.1 573 327 14.53 61.56 170.33 392.55 11.72 1.50

Radish 6.98 7.43 112.1 125.8 531 370 12.26 42.59 188.45 421.81 15.37 9.90

Cellulose+Radis

h 7.11 7.25 108.4 142.9 531 391 6.24 154.04 126.92

1229.3

1 20.33 7.98

PE+Radish 7.08 7.27 82.4 136.5 777 423 24.17 23.0 183.07 344.68 7.58 15.27

PS+Radish 6.95 7.28 114.5 117 702 391 9.14 86.50 177.50 620.24 19.42 7.17

Ecoflex+Radish 7.14 7.19 99.4 120.1 434 434 15.23 90.88 175.44 679.31 11.52 7.47


- 144 -

4.7.4. Statistics analyzes about Biplot PC1 versus PC2

Figures 4.51-4-52 are the statistics analysis of all parameters Principal Component Analysis

(PCA). The STATISTICA 6.0 software (StatSoft Inc., Tulsa, Oklahoma, USA) was used for

all statistical analysis. All numerical parameters before statistical analysis were normalized

and autoscaled: the result for each variable is a zero mean and an unit standard deviation

(Latorre et al., 1999).

Scatterplot (statistic undegraded log 12v*24c)

C t0

A t0

A-CE t0

A-PE t0

A-PS t0

A-E t0

C t1

A t1

A-CE t1

A-PE t1

A-PS t1

A-E t1

C t0

R t0

R-CE t0

R-PE t0

R-PS t0

R-E t0

C t1

R t1

R-CE t1

R-PE t1

R-PS t1

R-E t1

Ammonium

Nitrate

protease

phosphatase

heavy metal

pH

E.C

MI

-3,0 -2,5 -2,0 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 2,0

PC1

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

PC

2

Figure 4.51. Biplot PC1 Versus PC2.


- 145 -

Scatterplot (statistic undegraded log 13v*24c)

C t0

A t0

A-CE t0A-PE t0

A-PS t0

A-E t0

C t1

A t1

A-CE t1

A-PE t1A-PS t1

A-E t1

C t0

R t0

R-CE t0R-PE t0

R-PS t0

R-E t0

C t1R t1

R-CE t1

R-PE t1

R-PS t1

R-E t1

Ammonium

Nitrate

protease

phosphataseheavy metal

pH

E.C

MI

-3,0 -2,5 -2,0 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 2,0

PC1

-3

-2

-1

0

1

2

3

PC

3

Figure 4.52. Biplot PC1 Versus PC3.

The chemical and biological results were studied using the Principal Component Analysis

(PCA). PCA is a multivariate statistical data analysis technique which reduces a set of raw

data into a number of principal components which retain the most of the variance within the

original data in order to identify possible patterns or clusters between treatments and

variables. The PCs were extracted by applying the principal of main axis method. Only

component loadings >0.7 were considered for interpretation of the PCs. In PCA analysis, the

data are decomposed into separate sets of scores and loadings for each of the two modes of

interest (treatments and variables) and the whole variability of the data is explained in order

to provide a clear and more interpretable visualization of data structure in a reduced

dimension. In addition, PCA analysis gives information that can also be clearly presented

graphically (Carroll, et al. 2004).

In Tables 4.19-4.20 are reported the correlation analysis of all test parameters of two plants,

Oat (Avena sativa) and Radish (Raphanus sativus).

PCA analysis isolated three principal components (PC) (Total variance explained: 70.2%)

covering variables related to chemical and biochemical parameters of the different treatments

(Table 4.19). The highest loadings of the first PC (PC1, 24.2% of the total variance) included

the phosphatase activity and the metabolic index (DH-ase/WSC) which show positive

correlation (Figure 4.51), meaning that the carbon cycling was activated and was able to

sustain microbial activity. The second PC (PC2, 23.2% of the total variance) included the

ammonium and the protease activity, indicating the activation of nitrogen cycle. Finally, the

third PC (PC3, 22.8% of the total variance) included the nitrate and the electrical

conductivity demonstrating also that nitrate contributed to soil electrical conductivity, and

that the system is evolving toward the production of nitrate nutrients.


- 146 -

PC1 and PC2 were chosen for the interpretation of the biplot of scores and loadings (Figure

4.52), in order to draw conclusions about the effects of polymer materials application on the

soil chemical and biochemical characteristics.

The treatments, at T1 sampling time were shifted on the right of the biplot with respect to T0

and resulted located nearer to the biochemical parameters. This indicated a stimulation over

time of the phosphatase activity (Table 4.17) and metabolic index (Figure 4.50), which were

significant on PC1 axis.

On the contrary, the treatments at T1 were generally shifted towards negative values of the

second principal component (PC2), which represents the variation of ammonium and

protease activity, thus indicating a decrease of these parameters.

Considering the different treatments, PS and PE at T0 are located on the left negative side in

the biplot (PC1 axis) indicating a negative effect of these polymers on microbial metabolism.

After forty-five days (T1) incubation, such negative effects resulted less evident; the

treatments are, in fact, cluster in the positive side of the biplot.

Table 4.19. Principal Component Analysis (PCA) of Paremeters

PC1 PC2 PC3

Ammonium -0.220 0.881 -0.089

Nitrate 0.148 -0.291 0.866

protease 0.044 0.832 0.088

phosphatase 0.719 0.193 0.453

heavy metal -0.665 0.327 -0.105

pH 0.509 -0.117 0.410

E.C 0.012 0.384 0.813

MI 0.802 -0.008 -0.118

Variance explained (%) 24.2 23.2 22.8

Total variance explained: 70.2%)


- 147 -

Table 4.20. Correlation Analysis about Testing Paremeters of Oat (Avena sativa) and Radish (Raphanus sativus)

AVENA SATIVA

45 days WSC Amm Nitrate protease phosph Heavy-Me DH-ase pH E.C. M. Index Plant

biomass GI%

WSC 0.599 0.037 0.715 0.419 0.293 0.250 0.265 0.629 -0.569 0.405 -0.165

RA

PH

AN

US

SA

TIV

US

Amm -0.254 -0.535 0.977 0.755 0.761 0.076 0.504 0.406 -0.460 0.817 0.079

Nitrate -0.639 -0.314 -0.396 -0.028 -0.626 -0.268 -0.012 0.542 -0.299 0.259 0.519

protease 0.017 0.626 0.087 0.802 0.634 0.105 0.428 0.536 -0.522 0.758 0.172

phosph -0.226 -0.099 0.689 0.580 0.288 0.043 0.355 0.750 -0.425 0.623 0.450

heavy-Me -0.360 0.653 -0.365 0.024 -0,510 -0.214 0.774 0.023 -0.436 0.711 -0.533

DH-ase 0.023 0.358 0.231 0.95 0.822 -0.275 -0.483 -0.067 0.631 -0.697 -0.131

pH -0.801 0.155 0.271 -0.282 0.048 0.394 -0.143 0.423 -0.707 0.697 -0.468

E.C. -0.127 -0.154 0.364 0.313 0.817 -0.395 0.681 0.326 -0.667 0.636 0.178

M.Index -0.363 0.399 0.489 0.823 0.872 -0.135 0.922 0.163 0.691 -0.955 0.023

Plant

biomass -0.696 -0.777 0.851 -0.258 0.518 -0.714 0.054 0.384 0.393 0.292 0.206

GI% -0.579 -0.650 0.931 0.236 0.857 -0.511 0.444 0.196 0.657 0.673 0.670


- 148 -

The correlation coefficients among the parameters have been calculated separately for

Raphanus sativus and Avena sativa, to better relate soil metabolic processes and plant

germination and growth (Figure 4.50).

Raphanus sativus plant biomass correlated negatively with the WSC and ammonia, while it

was positively correlated with the nitrate. In the presence of this plant species, a high positive

correlation between metabolic index (MI) and protease and phosphatase activities was also

showed. On the contrary, in the presence of Avena sativa plant biomass was correlated with

ammonia and MI, but with the opposite trend. This confirmed again a more active

metabolism in Raphanus sativus than Avena sativa.


- 149 -

4.8. PHB biodegradation in soil

After one week from the treatments, total microbial cultivable population significantly

increased in both the PHB treatments (Figure 4.53) with respect to the untreated soil (B). The

highest dose showed the highest value. This trend is more evident when considering the

cultivable Pseudomonas genus (Figure 4.54). This means that the soil microorganism were

ready to utilize the PHB as a carbon source.

PHB-degrading microorganisms are ubiquitous in the environment and several extracellular

PHB depolymerases from Alcaligenes, Comamonas and Pseudomonas have been isolated,

purified and characterized (Yutaka Tokiwa, Buenaventurada P. Calabia 2004). PHB

depolymerases of Pseudomonas lemoignei hydrolyze PHB mainly into the dimeric and

trimeric esters of 3-hydroxybutyrate (Jendrossek, et al. 1993).

0,0E+00

1,0E+07

2,0E+07

3,0E+07

4,0E+07

5,0E+07

6,0E+07

7,0E+07

8,0E+07

B1 H1 L1

CFU

g-1

Total microbial population

Figure 4.53.Total Cultivable Microbial Population After One Week Incubation.

B, untreated soil, H, 2% of PHB in soil; L, 1% of PHB in soil.

0,0E+00

5,0E+04

1,0E+05

1,5E+05

2,0E+05

2,5E+05

B1 H1 L1

CFU

g-1

Pseudomonas

Figure 4.54. Pseudomonas Population After One Week Incubation.


The stimulation of microbial growth and activity was confirmed by the activation of the

arylesterase and PHB depolymerase activities (Figures 4.55, 4.56).

Arylesterase activity showed a significant increase after four weeks from treatments in both

PHB doses with respect to untreated soil, while the depolymerase was activated just after one


- 150 -

week.

This could mean that the two enzymes act consecutively on the PHB degradation:

depolymerase activity release ester fragments which are the substrate of other enzymes,

including arylesterase activity.

PHB can be degraded either by the action of intracellular or extracellular depolymerases

(Calabia and Tokiwa, 2006).

0,00

50,00

100,00

150,00

200,00

250,00

300,00

350,00

control soil high PHB Low PHB

mm

olP

NP

/Kg*

h)

Arylesterase activity1 week

3 week4week

Figure 4.55. Aryl Esterase Activity After One, Three and Four Week Incubation.

Control soil, untreated soil; high PHB, 2% of PHB in soil; Low PHB, 1% of PHB in soil.

Figure 4.55. PHB-Depolymerase Activity After One and Four Week Incubation.


Conclusion

- 151 -

5. CONCLUSIONS

The research work devoted to the accomplishment of the Doctorate Thesis can be divided in

two parts. The first was related to the assessment of processing profile, thermal properties

and biodegradation propensity for synthetic commercially available polyesters (Ecoflex and

Ecovio) and bio-based polymers (PHBV, PLA and their blends and composites).

The main conclusions of this part were, as follow, in details:

Biodegradation respirometric tests were carried out under different incubation

conditions, in the presence of microbial consortia present in different environmental

compartments such as forest soil, mature compost and river water.

Polymers biodegradation depends on the soil microorganisms activity and moisture

condition of the incubation medium.

Useful information on the effect of the composition of PHBV based materials on their

biodegradation behaviors have been collected.

It was confirmed that pure PHBV samples are readily biodegradable under all the

adopted test conditions.

Additive and fillers utilized in the preparation of the analyzed materials may affect

significantly the biodegradation kinetics in different environmental compartments.

The presence of the chain extender in a concentration of 5 percent by weight, which

was utilized with the aim to improve the mechanical properties of PHBV, induced a

negative impact on the PHBV biodegradation process.

The addition of ligno-cellulosic fillers can modify the negative impact of the chain

extender, thus increasing the biodegradation propensity as a function of its amount

inside the PHBV composite. The mitigating effect is more evident once the samples

were submitted to biodegradation in mature compost at 58°C.

The same evidences were noticed in the biodegradation experiments carried out in soil

& aqueous media.

The addition of a chain extender (Joncryl) in high concentration (5%) strongly affects

PHBV and PLA based composites biodegradation propensity in all analyzed media. On

the other side, a little concentration (0.2%) for this additive did not create a great impact

on the composites biodegradation behavior.

PLA and its blends and composites, in compost conditions, showed biodegradation

kinetics slower than those recorded for the analogous PHBV based blends and

composites.

Joncryl addition to neat PLA produced more stringent limits for biodegradation

propensity than those recorded for PHBV based samples.

The preliminary studies on PHBV processing profile showed that the suitable

concentration for the organic filler was 10%, for the inorganic filler was 3% and the

Joncryl amount was 0.2%. For PLA processing profile, the suitable concentration was

20% for lignin and 0.2% for Joncryl.

The addition of an organic ligno-cellulosic filler (powder obtained by the grinding of

hazelnut shells) alone or in combination with the chain extender Joncryl and the

inorganic filler (Dellite 72T) showed a reinforcing and compatibilizing action of these

components for PLA, as detected by the torque trend stability for the PLA/LN20/J5

composite compared to neat PLA.

Lignin and inorganic filler can significantly increase the onset temperature and residue

values for PHBV based composites. The composites become more thermally stable than

the neat polymer, thus they start their degradation at higher temperature. The results


- 152 -

obtained on PHBV/D3 composite show better thermal properties than PHBV/D6

composite.

PLA mixing with lignin, alone or in combination with a synthetic additive (Joncryl)

and/or an inorganic component (Dellite) produced a thermal stability increasing (Ton)

for the composites PLA/LN20 and PLA/LN20/J5.

PLA/Joncryl blends showed a complex DTGA trace, thus the chain extender promoted

cross-linking and oxidative reactions between epoxy –OH groups and the PLA end

capping OH groups.

Joncryl addition produced a molecular weight increase for PLA/Joncryl blends, as

detected by GPC analysis. Joncryl reactive sites selected preferably some polymeric

chains respect to others (double eluition peak), when its concentration did not exceed 2

percent by weight.

From DSC results, lignin and inorganic components can decrease the melting enthalpy

for the composites increasing experiment temperature.

A little amount Joncryl (0.2-2%) had no effect on the melting enthalpy, whereas a

significant decrease was observed by using a chain extender amount of 5 percent by

weight of addition produces a decrease of the enthalpy. PHBV/LN20/D3/J2 is the best

composite in terms of the lowest melting enthalpy.

The addition of Joncryl and Dellite produced a decrease in composites melting

temperatures, whereas lignin addition showed an increase of that parameter.

DSC thermograms, for PLA based composites, showed fairly high glass transition

temperature (Tg). That can be correlated with the rigidity and fragility of the composite

material.

The second part consisted of two experiments carried out at the Institute of Ecosystem Study

(ISE) of the National Research Council (CNR).

The experiment performed on the polymer materials PE, PS, Ecoflex and cellulose in a soil-

plant system, under controlled environmental conditions, showed that the selected

parameters were efficient and appropriate in discriminating the effect of the polymer

degradation on soil properties. In this experiment we selected several chemical and

biochemical parameters usually used to assess soil quality and its response to xenobiotic

compounds. The experiment has focused especially with a soil plant system to assess the

unbalance of nutrients and plant growth inhibition. Since PE, PS and Ecoflex represent three

different potential sources of available carbon, the water soluble carbon fraction of soil

organic matter (WSC) was studied in attempt to relate soil nutrient response to carbon

induced metabolism.

The biomaterial application increased the microbial metabolism in soil through the

supply of a potential carbon and nitrogen source.

The germination index (GI %) showed a negative effect of polymer application on plant

germination and growth only in the presence of Raphanus sativus, while Avena sativa

was not negatively affected. Therefore, in a short period, Raphanus sativus could be

considered as a most appropriate indicator of eco-toxicological effect of polymers. This

effect was consolidated by an appropriate statistical treatment, which clearly indicated

that the polymers promoted the carbon cycle and in turn activated organic nitrogen

metabolism, bringing the soil plant system towards the nitrate nutrient production.

Ecoflex compared to PE and PS showed the higher nitrate/ammonia ratio and the lower

water soluble carbon content (WSC). This indicated that the system prevents the loss of

carbon and the potential surface and ground water pollution, while maintains an

equilibrate level of available nitrogen nutrients.

In the preliminary experiment of the PHB polymer biodegradation, specific

Conclusion

- 153 -

microorganisms and enzyme activities were investigated. Total microbial cultivable

population and Pseudomonas genus significantly increased in both high (H) and low (L)

PHB treatments with respect to the untreated soil. The stimulation of microbial growth

and activity was confirmed by the activation of the arylesterase and PHB depolymerase

activities. The very high stimulation of Pseudomonas genus, particularly at the high

dose of PHB (2%), suggested its involvement in the PHB degradation through the

synthesis of the depolymerase enzyme. This preliminary approach must be better

investigated in details with appropriate experiments where the enzymes are extracted

from soil and purified in order to plan efficient enzyme-assisted processes of polymers

biodegradation in a real environment.


- 154 -

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