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Towards a Greener Future

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004171/00001

Material Information

Title: Towards a Greener Future GC-MS Analysis of Poly-3-Hydroxybutyrate (P3HB) Content in PANICUM VIRGATUM.
Physical Description: Book
Language: English
Creator: Seuraj, Adesh
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2009
Publication Date: 2009

Subjects

Subjects / Keywords: Poly-3-hydroxybutanoate
Biodegradable Plastics
Butanolysis
Biopolymers
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Plastic production has increased markedly over the last hundred years. With greater consumption rates today, than ever before, serious attention to the consequential and fringe effects of the plastic industry has provoked the scientific interests of ecologically minded individuals. A non-polluting alternative is the use of plants to produce plastics that are biodegradable, easily commercialized, and specialized for aesculapian applications. In the formulation of a more benign product, the performance integrity and properties of traditional plastics remains uncompromised. Butanolysis is a primary screening technique for the presence of poly-3- hydroxybutyrate in the early life cycles of a plant. At Metabolix, (Cambridge, MA.), extensive analysis of transgenic plant samples revealed five batches containing P3HB quantities in targeted ranges for economic competitiveness with traditional synthetics. Of the five batches, the most promising samples were forwarded to the engineering team for advancing the P3HB biofactory pathway, and improving the efficiency of downstream commercialization.
Statement of Responsibility: by Adesh Seuraj
Thesis: Thesis (B.A.) -- New College of Florida, 2009
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Scudder, Paul

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2009 S4
System ID: NCFE004171:00001

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004171/00001

Material Information

Title: Towards a Greener Future GC-MS Analysis of Poly-3-Hydroxybutyrate (P3HB) Content in PANICUM VIRGATUM.
Physical Description: Book
Language: English
Creator: Seuraj, Adesh
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2009
Publication Date: 2009

Subjects

Subjects / Keywords: Poly-3-hydroxybutanoate
Biodegradable Plastics
Butanolysis
Biopolymers
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Plastic production has increased markedly over the last hundred years. With greater consumption rates today, than ever before, serious attention to the consequential and fringe effects of the plastic industry has provoked the scientific interests of ecologically minded individuals. A non-polluting alternative is the use of plants to produce plastics that are biodegradable, easily commercialized, and specialized for aesculapian applications. In the formulation of a more benign product, the performance integrity and properties of traditional plastics remains uncompromised. Butanolysis is a primary screening technique for the presence of poly-3- hydroxybutyrate in the early life cycles of a plant. At Metabolix, (Cambridge, MA.), extensive analysis of transgenic plant samples revealed five batches containing P3HB quantities in targeted ranges for economic competitiveness with traditional synthetics. Of the five batches, the most promising samples were forwarded to the engineering team for advancing the P3HB biofactory pathway, and improving the efficiency of downstream commercialization.
Statement of Responsibility: by Adesh Seuraj
Thesis: Thesis (B.A.) -- New College of Florida, 2009
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Scudder, Paul

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2009 S4
System ID: NCFE004171:00001


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TOWARDS A GREENER FUTURE: GC-MS ANALYSIS OF POLY-3HYDROXYBUTYRATE CONTENT IN PANICUM VIRGATUM. BY ADESH SEURAJ A THESIS Submitted to the Division of Natural Sciences New College of Florida in partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Dr. Paul Scudder Sarasota, Florida June, 2007

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ii Dedication For you*. It is, after all, your prerogative. *Daughtry, Alfano, Dookera n, Millington & Scudder.

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iii Adesh Seuraj New College of Florida, 2007 Prologue. Scientific advancement has always been fueled by the need to question present understandings and beliefs. It is only when we are critical of what we have, can we possibly decide where we could be The nature of this thesis is essentially that. It entails improvement of a commodity that man has become almost unquestionably reliant upon: plastic. Since its discovery at the turn of the 18th century, the plastics industry has become one of the worlds most booming enterprises. Plastics possess properties that make them one of the most sought after and versatile comm odities for everyday life. But herein lies a dilemma. This thesis attempts to illuminate this dilemma and make recommendations for a comparable, more sustainable, and environmentally benign product. As the title suggests, the thesis is com posed of two parts. Th e first addresses the ecological state of the planet, and is emblem atic of the need to curb the cycles of pollution that are so erosive to the biosphere. In our considerations of bio-based plastics, it is but one factor in a circ uitous maze of complexity, func tionally capable of restoring order to biogeochemical cycles. The first seven chapters of the thesis follow a natural progression from oil based synthetics, through the biopolymers, and finally the bioplastics. This order is im portant and it gives the thesis a clear sense of direction. Chapter one opens with a clarification of traditional plastics and bioplastics, making an early distinction between them. Synthetic pol ymer chemistry as well as the methods of processing plastics, including additives, will be discussed throughout the remainder of the

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iv chapter. Finally, a brief section on step-g rowth and chain growth polymerization is included, for its foreseeable helpfulness in future sections, where methods of degradation, including fragmentation and hydrolysis will be addressed. While chapter one differentiates plastics ba sed on their source of origin, chapter two further categorizes them based on their behavi or when subjected to heat. Thermoplastics and thermosets will be illustratively explaine d, and examples and applications of each will be given. These first two chapters can be envisaged as dealing with plastics at their prime. Chapter three does the opposite and engages a consideration of what happens after? Methods of plastic degradati on will be described as well as the environmental factors that encourage biodegradation. Three idealized situations ba sed on the performance index of a plastic will be considered as far as appl ication specificity is concerned. Chapter three concludes with the need for a benign alternativ e, and preludes the essence of chapter four, where biopolymers are extensively reviewed fr om a then and now perspective. This is natures chapter, and natural fibers and composites are discussed alongside the three main biopolymer groups. In addition, the structural compositions of biopolymers in relation to their functionality elucidates the highly de veloped and sophisticated systems present in nature. From the insight gained from biopolymer s, there is a natural transition to chapter five: Bioplastics. The history of bioplastics will be addre ssed, as far back, when it was nothing more than an entrepreneurial curiosity to the pres ent day, where re-emergence of efforts in this field is abundant in numerous industries. In addition, current methods of biopolymer production via fermentation and agronomic en deavors will be focused upon. In chapter

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v six, the importance of bioplas tics in the medical field wi ll be underscored and poly-4hydroxybutyrate (P4HB) will be compared to the biodegrad able synthetic polymer, polyglycolic acid (PGA) in applications like car diovascular surgeries, and graft cogitated redevelopment. After having been sensitized to the need for, and benefits of, a green alternative, chapter seven will show that what is justified in principle is not botched in practice. The experimental section describe s a process known as butanolysis, developed at Metabolix, (Cambridge, MA.), and used to analyze th e polymeric content of transgenic, PHA producing plants, using Gas Chromatograp h Mass Spectrometry(GC-MS). Routine analysis of thousands of batc hed switchgrass samples yielded five batches with promising results. These batches were labeled batc h 10, batch 11, batch 12, batch 13, and batch 14. The results are recorded in a write-up, which takes the form of a standard laboratory report. The results from batches 10 through 14 will be discussed in light of the adjudged feasibility of using switchgrass as a PHA producing plant. The discussion section will describe the entire process of PHA producti on in switchgrass as we ll as the economic targets that must be achieved to make this process feasible. Future work aimed at improvisation will also be mentioned. In conclusion, there is a difference betw een towards a greener future and to a green future. Succinctly put, the former is a prelude to the latte r. Towards has the implication that a movement is being made in some general directi on. It lacks specificity and is indicative of the conception stages of a project. At present, this thesis embodies a progression towards a greener future. The even tual, admirable aim, is of a green one.

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vi TABLE OF CONTENTS Dedication ii Prologue iii Table of Contents vi Abstract ix Chapter 1 TRADITIONAL PLASTICS 1 1.1 Distinction between traditional plastics and bioplastics. 1 1.2 How plastics are used. 1 1.3 Synthetic polymer chemistry. 2 1.4 Polymer chemistry. 3 1.5 Two types of polymerization. 5 1.6 Additives. 7 1.7 Processing Methods Extrus ion, Injection molding, 9 Compression molding, Blow molding, Transfer molding, Vacuum forming, Rotational molding. Chapter 2 TWO CHARACTERIZATIONS OF PLASTICS 13 2.1 Thermosets Polyurethanes, Unsaturated Polyesters, 13 Epoxies 2.2 Thermoplastics Polyet hylene, Polypropylene, 15 Polyvinyl chloride Chapter 3 PLASTIC DEGRADATION 18 3.1 Nondegradable plastics 18 3.2 Readily degradable plastics 19

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vii 3.3 Programmed degradable plastics 20 3.4 BiodegradationNatures recycling. 22 3.5 Factors affecting the rate of biodegradation 23 3.6 Degradation considerations 24 3.6 (a) Plastics degrade by many routes. 24 3.6 (b) The chemical and structural composition of the 25 plastic greatly affects the degradation rate. 3.8 The need for an alternative. 27 Chapter 4BIOPOLYMERS 29 4.1 History of Biopolymers 29 4.2 Carbohydrates 4.2(a)Cellulose 30 4.2(b)Starch 31 4.2(c)Chitin 31 4.3 Proteins 32 4.3(a)-Collagen 33 4.3(b)-Casein 33 4.3(c)-Whey 33 4.3(d)-Plant protein 33 4.3(e)-Amino Acids 34 4.4 Polyesters 34 4.5 Natures fibers 4.5(a)-Cotton and Wool 36

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viii 4.5(b)Silk 36 4.6 Natures composites 37 Chapter 5BIOPLASTICS 5.1 Introduction to Bioplastics 39 5.2 Early Bioplastics 40 5.2 (a)Keratin and Gelatin 40 5.2 (b) Casein 41 5.2 (c) Soybeans 41 5.2 (d)Ebonite & Gutta percha 42 5.2 (e)Cellulose nitrate 43 5.2 (f) Cellulose acetate 44 5.2(g) Rayon and Cellophane 44 5.3 The New Bioplastics 45 5.4 Biopolymers extracted directly from their natural origin 46 5.4 (a)-Carbohydrates starch 46 5.4 (b)-Chitin and Agar 48 5.4 (c)Proteins (Soy, Zein and Gelatin) 48 5.5 Biopolymers produced from fermentation 49 5.5 (a) Polyesters produced from bacteria 49 5.5 (b) Lactic Acid Fermentation 50 5.6 Triglycerides 51 5.7 Biopolymers produced from plants 52 5.8 Biodegradable synthetic polymers 53

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ix Chapter 6 MEDICAL APPLICATIONS OF PHAs 54 6.1 Production 54 6.2Mechanical properties 55 6.3 Processing 56 6.4Sterilization 57 6.5Biocompatibility 57 6.6Absorption 58 6.7Applications 59 6.7 (a) Congenital cardio vascular defects artery augmentation 59 6.7 (b) Heart valves 61 6.7 (c) Sutures, medical textile products and bulking agents. 62 6.8 Conclusion 62 Chapter 7 ANALYSIS OF P3HB CONTENT IN PANICUM VIRGATUM 64 7.1 Butanolysis 65 7.2 Introduction 65 7.3 Experimental Procedures and Methods 66 7.4 Calculation considerations for GCMS analysis 67 7.4 Results 69 7.5 Discussion 84 7.5(a) Chemistry of Butanolysis, a nu cleophilic substitution reaction. 84 7.5(b) Sources of Experimental Error 85 7.5(c) History of PHA: Bacteria l PHA Biosynthetic Pathways. 88 7.5(c) Economics of PHA development in plants 89

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x 7.6 Microbial gene expression 89 7.6(a)Cytoplasmic expression 90 7.6(b)Plastid expression 90 7.7 Putting switchgrass in context 92 7.8 Conclusion 93 References 94 Diagrams and Table of Figures 98

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xi List of Figures Page number Figure 1.1-How plastics are used in the United States. 2 Figure 1.2Arbitrary monomer A combining to produce 5 discreet oligomers, a nd indiscreet polymers. Figure 1.3 Condensation/step growth polymerization to form PET. 6 Figure 1.4 Mechanism of addition/ chain growth polymerization to form polyethylene. 7 Figure 1.5Extrusion molding through a die to form extruded product. 9 Figure 1.6 Injection molding device. Figure 1.7Compression molding de vice with "charge" confined between the male and female mold halves. 11 Figure 1.8 Process of blow molding. 11 Figure 1.9 Stages in transfer molding. 11 Figure 2.1 Structural differen ces between thermoplastics and thermosets. 13 Figure 2.2 Epoxy prepolymer form ed from epichlorohydrin and bisphenol-A. Figure 2.3 Structural formula of polyethylene 16 Figure 2.4 Polymerization of vinyl chloride to form PVC. 17 Figure 3.1 Degrade curve of a non-degradable plastic. 19 Figure 3.2 Degrade curve of a readily degradable plastic 20 Figure 3.3 Degrade curve of a pr ogrammed degradable plastic 22

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xii Figure 4.1Structural formula of cellulose, with 1-4 linkages. 31 Figure 4.2Structural fo rmula of starch, with 1-4 linkages. 31 Figure 4.3 Structural fo rmula for a protein. 32 Figure 4.4Chemical structure of major PHAs. 35 Figure 5.1 Trans polymer of gutta percha. 42 Figure 5.2 Cellulose nitrate polymer. 43 Figure 5.3 Cellulose acetate polymer. 44 Table 5.1 Biopolymers that can be extracted directly from their natural environment. 50 Figure 5.4Polymerization of lactic acid to form polylactic acid (PLA) using dimeric ring-opening Table 5.2Biopolymers obtained indi rectly from natural sources 52 Figure 5.6 Some biodegradab le synthetic polymers 53 Table 6.1 Comparison of P3HB, P4 HB and their co-polymers. 61 Table 6.2 -Comparison of physical and chemical properties of P4HB and PGA 60 Figure 7.1Standardized curve employi ng butanolysis to find [P3HB] 69 Table 7.2Highest producing P3HB samples by batch 71 Figure 7.2 Diagrammatical co mparison/representation of batch frequencies. 71 Figure 7.3 -Target ions for dipheny lmethane internal standard. 72 Figure 7.4 -Target ions fo r butyl-3-hydroxybutyrate 73 Figure 7.5-Standard curve for batch 10 74

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xiii Table 7.3Data for batch 10. 75 Figure 7.6-Standard curve for batch 11 76 Table 7.4Data for batch 11. 77 Figure 7.7-Standard curve for batc h 12 of Amount Ratio versus Response Ratio 78 Table 7.5Data for batch 12 79 Figure 7.8-Standard curve for batch 13 80 Table 7.6Data for batch 13 81 Figure 7.9-Standard curve for batch 14 82 Table 7.7Data for batch 14. 83 Figure 7.10 Mechanism of Butanolysis. 85 Figure 7.11Pathway of PHB synthesis in A. Eutropus 88 Figure 7.13 Modification of plant metabolic pathways for the synthesis of P3HB and P3HB3HV 91 7.14 Accumulation of P3HB granule inclusions in chloroplasts of A. thaliana 92

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xiv TOWARDS A GREENER FUTURE: GC-MS ANALYSIS OF POLY-3HYDROXYBUTYRATE (P3HB) CONT ENT IN PANICUM VIRGATUM. Adesh Seuraj New College of Florida, 2007 Abstract Plastic production has increased markedly over the last hundred years. With greater consumption rates today, than ever before, se rious attention to th e consequential and fringe effects of the plastic industry has provoked the scientif ic interests of ecologically minded individuals. A non-polluting alternative is the us e of plants to produce plastics that are biodegradable, easily commercia lized, and specialized for aesculapian applications. In the formulation of a more benign product, the performance integrity and properties of traditional plastics remains uncompromised. Butanolysis is a primary screening technique for the presence of poly-3hydroxybutyrate in the early life cycles of a plant. At Metabolix (Cambridge, MA.), analysis of thousands of tran sgenic plant samples revealed five batches containing P3HB in targeted ranges for economic favorability. Of the five batches, the most promising samples were forwarded to the care and e xpertise of the plant engineering team for advancing the panicums potential as a biofactory for P3HB production. ____________ Dr. Paul Scudder Natural Sciences

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1 CHAPTER 1 Traditional plastics. 1.1 Distinction between traditional plastics and bioplastics. Plastic: Any of various complex organic compounds produced by polymerization, capable of being molded, extruded, cast into various shapes and films, or drawn into filaments used as textile fibers. -Webster's Dictionary The above definition does little in differentiating between traditional plastics and bioplastics, of which clarity should be of ut most importance. For the purposes of this thesis, the term traditional plastics will refer to polymers derived from petroleum, whereas the term bioplastics will refer to those derived fr om renewable plant sources and microorganisms. 1.2 How plastics are used In 1986, forty three billion pounds of plastic were produced. Fourteen years later, this value doubled.1 A growing population was culpable for this increase, and in spite of a declination of reserves, high demand offset the theoretical price increase of the commodity. In the United States, the use of plas tics can be represented in the pie chart in Figure 1.1.

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2 Figure 1.1-How plastics are us ed in the United States. The largest section of the pie chart, approximately 30%, is used of packaging. Packaging can be categorized as either primary, secondary an d transit. Primary packaging will be the most familiar to the average consumer, and includes sandwich wrappings, plastic bottles and food containers. This packag ing is always in dire ct contact with the food and represents the first line of protection from external hazards.2 Secondary packaging, like crates and larg er cases, may be used to group several primary packaged products for transportation. A plastic bottle filled with water may be primary packaged, but twenty four of them, are held in a secondary packaged crate. Wholesale grocery and membership clubs are known for the vast amounts of secondary packaging used for bulking individual products. Transit packaging, sometimes called tertiary packaging, is used to ensure the safe handling and transportation of packaged produc ts, whether primary or secondary. Shrink wrapped pallets and corrugated cartons are quite useful in reducing the vibrations and

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3 compressions encountered by products during deli very from manufacturer, to distributor, to consumer. Building and consumer products, together represent an equivalent area on the pie chart as packaging, while furniture and electrica l applications represent the smallest area of plastic usage in the United States. Th is chart bears close resemblance to the compartmentalized usage in the United Ki ngdom, and worldwide, there are minor fluctuations in overall category percentages. 1.3 Synthetic Polymer History Polymer chemistry can be broadly di vided into biological and non-biological macromolecules. In this section, only the non-biological polymers will be discussed. Synthetic polymers are a class of organi c molecules synthesi zed by man-directed initiatives under controlled c onditions of temperature, ca talysts and other reagents. Economically, the importance of the synthe tic polymer field is exemplified by the recruitment of one third of all American ch emists and engineers to work in this, and similar industries.3 Discovery of the first synthetic polymer was by Leo Bakela nd in 1907. He called it bakelite. After its discovery, active resear ch in the development of polymer chemistry began with the production of vinyl chloride resins in 1927. Th ree years later, polystyrene was invented as well as its expanded form, styrofoam.4 On account of good insulating properties, and its light weight, it immediatel y became a very marketable item. In 1938, Wallace Carothers Dupont produced nylon, the fi rst synthetic polymeric fiber. Three years later, one of the most popular synthetics was discovere d. It was called polyethylene

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4 and it became the most popular plastic in the world on account of its variable density and wide range of applications. Section 1.4 introduces the chemis try of polymer synthesis via polymerization reactions. 1.4 Polymer Chemistry. Organic materials are based on polymers Polymers can be formed from the conversion of natural products or by synthesi s from primary chemicals called fossil fuels. Fossil fuels are composed of elements such as carbon (C), hydrogen (H), nitrogen (N), chlorine (Cl) and sulfur (S). Crude oil is one such fossil fu el, and is extracted using offshore oil rigs. Crude oil is fr actionally distilled, and separa ted into fractions, based on differing boiling points, for a wide variety of applications. The desired fraction for the petrochemical industry lies between the petrol and naphta range. The most popular intermediaries are methane, ethylene, propy lene, and butylene. These feedstocks are called monomers. When monomers join repeatedly a polymer ization reaction occurs and large, high molecular weight polymers are formed. A substance that cons ists of a finite number of monomers joined together is called an oligomer while one that consists of an indeterminate amount is called a polymer. Oligomers have a discreet, known degree of polymerization whereas polymers do not. Since monomers are variable due to the atoms they contain as well as their structural ar rangements, polymers are equally as variable. Consider the arbitrary monomer, A in Figure 1.2.

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5 Monomers: A A A A Oligomers: A-A-A-A (tetramer) A-A-A (trimer) A-A (dimer) Polymers: A1-A2-A3--A or, for simplicity, -[A-A]nFigure 1.2Arbitrary monomer A combining to produce disc reet oligomers, and indiscreet polymers. When monomers polymerize, the net re sult is essentially a high molecular weight polymer called a resin If the same monomers combine, homopolymer resins are produced and if different monomers combine copolymer resins are produced. Resins may be processed into different shapes and ge ometries using different techniques. Sheets, films, and three dimensional objects can be ma de for specific market applications. Before processing occurs, additives must be included to enhance the properties of the resin for the desired application. A comm on example would be a baker who combines butter in his dough, before baking, to enhance the texture of the bread. 1.5 Two Types of Polymerization. There are two types of polymerization reactions that monomers may undergo. The first is addition polymerization, where the m echanism is usually referred to as chain growth. The second is condensation polymeri zation where the mechanism is referred to as step-growth. It is usef ul to bear in mind that there are exceptions to these classifications, but nonetheless it is a good rule of thumb. Condensation Polymerization A chemical reaction in which a small molecule separates from two or more monomers upon their combination to form a polymer. Mechanistically, polymers are formed where both ends of the growing ch ain have functional groups which can react

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6 with a monomer or with the appropriate functional group on another chain. See Figure 1.3. Figure 1.3 Condensation/step grow th polymerization to form PET. Addition Polymerization A chemical reaction in which simple mono mers are added to each other to form polymers without by-products. Mechanistica lly, the monomers become part of the polymer chain by sequential addition, one at a ti me. In most cases it is radical initiated. Figure 1.3 shows the mechanism of radical initiated addition, for the conversion of ethylene to polyethylene. The first stage i nvolves the generation of an alkyl radical formed by the homolytic fission of a covalent bond. This radical can subsequently attack an ethylene molecule to generate another radical (R-CH2-CH2*). A chain reaction thus develops, and the (R-CH2-CH2*) radical may add to an indeterminate number of ethylene molecules to generate a polymer.

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7 Figure 1.4 Mechanism of addition/chain growth polymerization to form polyethylene. 1.6 Additives After the polymer resins have been produced by polymerization, additives must be incorporated into the formula tion. Without additives, the poly mers are of little practical use, possessing minimal properties for product application. Most po lymers are blended with additives during processing into their finished parts. Additives are incorporated into polymers to alter and improve their basic m echanical, physical and chemical properties. The net result of mixing polymers w ith additives is called compounding.5 The main types of additives are: Process additives These are important during processing. Process additives are used as lubricants and viscosity boosters, as well as foaming agents for styrofoam cups. Such additives make processing easier and also saves money, sin ce heat damage to the plastics can be minimized and molding temperatures can be lowered. In other cases, they are

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8 indispensable to the formation of the final pr oduct as in the styrof oam cups, previously mentioned. Stabilizers Stabilizers increase product lifespan and gi ve the plastic resistance to the degrading effects of sunlight, chemicals and bacteria. For outdoor applications, antioxidants are very useful for protecting the plastic under hostile conditions. Performance additives These are added to the plastic to increase the safety and durability of the final product. Such additives may be reinforcing agents like glass fibers or things like colorants. More important applications incl ude impact modifiers, flame retardants and anti-static agents. Plasticizers These are softening agents used to control the flexibility and rigidity of the polymer. They work by loosening the polymer and facilitating motion between the different segments. The majority of plasticizers are us ed for polyvinyl chloride (PVC) in diverse applications such as coatings, plumbing, cons truction materials, credit cards and plastic bottles. Phthalates are the most widely used plasticizers, and in PVC, for example, dioctyl phthalate (DOP) is an important additive. Phthalates are often crucial to a products application but are difficult to localize for a specific period of time. This is the main drawback of phthalate usage.

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9 When brand new, the distinctive smell in side the automobile can be attributed to the volatile phthalates. Over time, phthalates can migrate to the surface of the plastic, and evaporate or leach into the surrounding envi ronment. Eventually, there is near total migration into the environment and the plasti c becomes brittle. Such is the case of an aging dashboard in an old automobile. The release of phthalates into the envir onment also poses environmental and other health related concerns. As a result of their worldwide abundance, phthalates have become a major industrial pollutant. In resp onding to this predi cament, efforts into deriving more benign plasticizers from vegetable oils have been successful. Currently, vegetable oil plasticizers account for 15% of the total US market for plasticizers6. 1.7 Processing Methods At this point, the final product, usually in solid form, is ready to be processed into three dimensional shapes or sheets of film. During processing, the plastic resin can exhibit many degrees of plasticity and the end product may be quite hard. There are a variety of different processing methods used to convert resi ns into finished products. Some include: Extrusion A process in which heated or unh eated plastic is forced through a die (shaping orifice) in one continuously formed shape. Either a mechan ical or a hydraulic

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10 press may be used. It is analogous to a pasta makers operation. Typical parts produced by extrusions are trim parts used in automotive and construction applications.6 Figure 1.5Extrusion molding through a die to form extruded product. Injection Molding This is the process of forming a material by forcing it, in a fluid state under pressure, into the cavity of a closed mold. The plastic material is placed into a hopper which feeds into a heating chamber. A plunger pushes th e plastic through the heating chamber where the material is softened and then forced into a closed mold. After the plastic cools to a solid state, the mold opens and the finished product is ejected. This is mainly used for thermoplastics but to a lesser extent, thermosets. blank die

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11 Figure 1.6 Injection molding device. Resin formulati on is placed in hopper, softened by heating, force injected into mold, then ejected as finished product. This processing method is quite popular for thermoplastics. Compression Molding This involves molding a materi al already in a confined cavity by applying pressure and usually heat. It is quite effective for polymer sheeting. The moldable material is referred to as a charge of sheet molding compound7 and is nestled between the male and female mold halves. The fe male half is concave shaped and receives the charge from the male half, which is slightly more c onvex shaped, with the charge located on its surface. Compression molding is mostly used to make larger flat or moderately curved parts such as hoods, fenders, scoops, spoilers, lift gates and the like for automotive enduses.

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12 Figure 1.7Compression molding device with "charg e" confined between the male and female mold halves. A combination of heat and pressure is used to create the final product. Mainly used for thermosets Blow Molding The main use of this technique is in th e manufacture of plastic drinking bottles and it affords us the advantage of producing hollow shapes without having to join separate parts. Blow molding is a pro cess used in conjunction with extrusion. A heated plastic mass is forced into the shape of a chilled mo ld cavity using compressed air. The goal is to obtain an even melt, form it into a tube with a desired cross secti on and blow it into the exact shape of the product. See Figure 1.8. Figure 1.8 Blow molding. Preform is manually stretched, and blown to take the shape of the mold. Transfer Molding A method of forming products by fusing a pl astic material in a chamber and then forcing essentially the whole mass into a hot mold where it solidifies. One of the main

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13 advantages of transfer molding is that it provides better product consistency and shorter cycle times than compression molding. Figure 1.9 Stages in transfer molding. (1) Resin is placed in reservoir containment. (2) and (3) Pressure is applied and the resin is forced into the mo ld. (4) pressure is relea sed, and top half of the mold is opened to recover the molded charge. Vacuum Forming A forming process whereby a heated plasti c sheet is drawn against the mold surface by evacuating the air between it and the mold. The vacuum forming process can be used to make most product packaging, speaker casings and even car dashboards.8 Rotational Molding In this process, heat is used to melt a nd fuse a resin inside a closed mold in the absence of pressure. It consists of a mold mounted on a machine cap able of rotating on two axes simultaneously. Solid or liquid resin is then placed within the mold and heat is then applied. Rotation distri butes the plastic into a uniform coating on the inside of the mold until the plastic part cools and sets. This process is used to make hollow, spherical configurations. This is used because polymer s are poor thermal conductors and heat is not transmitted through the polymer melt layer as readily as through the metal mold.9

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14 CHAPTER 2 Two Characterizations of Plastics 2.1 Plastics behave differently when subjected to heat. There are two characterizations of plasti cs based on the behavior of their resin when subjected to heat. Depending on the characterization, their properties and applications may vary widely, as well as their met hod of processing. Figure 2.1 describes the structural and thermodynamic differences between thermosets and thermoplastics. Figure 2.1 Structural differences between therm oplastics and thermosets. Note that thermosets possess cross links, whereas thermoplastics do not. When subjected to heat thermoplastics melt, but thermosets degrade. 2.1

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15 Thermoset A thermoset is a polymer that solidifies or "sets" irreversibly when heated. It is analogous to boiling an egg. When cooked, an egg solidifies and cannot reversibly become uncooked and soft. Thermosets are valu ed for their durabilit y and strength and are used primarily in automobiles and construc tion. The structures of these plastics bear high degrees of cross linkages in which the pol ymer chains are chemically interconnected with bridging groups forming a large three dimensional network. Bakelite, the first synthetic polymer is a thermoset. It was produced by the polymeri zation of phenol and formaldehyde.10 Other examples of thermoset plastics and their product applications are: Polyurethanes Polyurethanes are a class of polymers that are made by reacting diisocyanates (organic compounds containing two functional groups of structure -NCO) with dialcohols (a molecule with two or more hydroxyl groups ). They are a class of extremely versatile polymers that are made into flexible and rigid foams, fi bers, elastomers, and surface coatings. Common applications include mattr esses, cushions, insulation, ski boots, and toys. Unsaturated Polyesters These are linear copolymers that contain th e -COOfunctionality. The copolyesters are prepared from a dicarboxylic acid or its anhydride (usual ly phthalic anhydride) and an unsaturated dicarboxylic acid or anhydride, along with one or more dialcohols.11 They are used in lacquers, varnishes, boat hulls and furniture.

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16 Epoxies Epoxies are polyethers built from monomers, in which the ether group takes the form of a three-membered ring known as the epoxide ring. Most common epoxy resins are produced from a reaction between epichlorohydrin and bisphenol-A These two monomers first form an epoxy prepolymer th at retains two terminal epoxide rings. The prepolymers are further polymerized through the opening of the terminal epoxide rings by amines or anhydrides. This process, called curing yields complex, thermosetting network polymers in which the repeating units are linked by linear ether groups. They are used in glues, adhesives and in structur al parts such as laminated circuit boards.11 Figure 2.2 shows the structural formula of an epoxy prepolymer, as well as the product of amine polymerization of the prepolymer.

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17 Figure 2.2 Epoxy prepolymer formed from epichl orohydrin and bisphenolA, followed by amine polymerization of the prepolymer. 2.1 Thermoplastic A thermoplastic is a polymer which can be repeatedly softened by heating and hardened by cooling. In the softened state it can be shaped by mold ing and extrusion. The system is analogous to the state change water undergoes. Ice will melt when heated, but will readily solidify when cooled. This process is reversible and can be repeated many times. The structures of these plastics are such that they are mostly linear and unbranched. Examples of thermoplastics are: Polyethene

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18 This is produced in many forms having different densities. De nsity reflects the percentage of crystallinity, an important factor in determin ing mechanical properties. The less branched the polymer, as well as the more effective the packing in the crystal structure, the greater the tensile strength it has. For example, high-density polyethylene (HDPE) has a low degree of branching, and thus stronger intermolecular forces and tensile strength. HDPE is used in milk jugs water pipes and detergent bottles. Medium density polyethylene (MDPE) is typically used in shrink film and packaging film. Figure 2.3 shows the structural formula of polyethylene. Figure 2.3 Structural formula of polyethylene. Low density polyethylene (LDPE) has a high degree of short and long chain branching, which means that the chains do not pack into the crystal structure as well. LDPE has less tensile strength than MDPE, but is more ductile. LDPE is created by free radical polymerization It is used in plasti cs bags and film wraps. For common commercial grades of medium -density and high-density polyethylene, the melting point is typically in the rang e 120-130C. The melting point for average commercial low-density polyethylene is typica lly 105-115C. It should be noted that HDPE and LDPE form the ends of the clas sification spectrum based on density. There are many other forms of polyethylene, like li near low density polyethylene (LLDPE) that is beyond the scope of this thesis.

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19 Polypropylene Commercial polypropylene has a level of cr ystallinity intermediate between LDPE and HDPE. As a result of this median modulus its tensile strength is less than HDPE, and its flexibility is less than LDPE. It is, howev er, much less brittle than HDPE and can be used for luggage, molded automobile parts, fish nets, and appliance parts. Polypropylene also has uses in the packag ing industry and is quite common in dairy containers. This polymer ha s very good resistance to fatigue so that most plastic living hinges such as those on flip-top bo ttles and tic-tac lids are made from this material. Polyvinyl chloride (PVC) PVC is made by polymerization of the alkyl halide, vinyl chloride, and is produced in both flexible and rigid forms. When flexible, PVC is used in the processing of film and sheeting, and for packaging applications. Ot her uses include floor and wall coverings, when it is referred to as vinyl. Rigid PVC is actually unplasticized polyvinyl chloride (u PVC). It is used in the building industry for gutters, downpipes and window frames. Figure 2.4 shows an equation based on structural formula for the polymerization of vinylchloride to form polyvinyl chloride.

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20 Figure 2.4Polymerization of vi nyl chloride to form PVC. CHAPTER 3 Plastic Degradation The environmental degradation of plastics is not a simple process. The details of how plastics degrade after disposal are not tota lly understood and is th e subject of current research. It would behoove the reader to consider three idea lized cases of a degradable plastic, at the point of dispos al. The first case characterizes a plastic that is resilient to degradation. The second case characterizes one that is readily degr adable, and the third, one that is intermediate between the first two extremes. 3.1 Non-degradable Plastics Initially, plastics were manufactured to be long-lasting and resist degradation. There was nothing to suggest eventual problems of post usage disposal, as well as other impacts to non-renewable resources and the environm ent. The main concern was that these plastics were highly stable and served their functions well. Their pe rsistence for the most part, originated in three of their proper ties that made them so useful for many applications. They were strong, water resi stant and unattacked by microorganisms. An idealized, plastic degradation curv e, is drawn with ti me on the x axis and performance integrity(P.I) on the y axis. The performance integrity measures the stability

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21 of the plastic and declines either abruptly, or slowly as the plastic loses its strength and disintegrates.12 In Figures 3.1 through 3.3, the blue dot indicates the point at which the plastic is discarded, i.e. after it has outlived its useful life. Figure 3.1 shows a degradation curve of a plastic that does not measurably degrade on any reasonable time scale. It persists for a long period, even after it is deemed unfit for further usage. Non-degradable plastics approximate many current comm odity plastics, like the polyolefines: polyethylene, polyvinyl chloride polypropylene, and polystyrene.13 Figure 3.1 Degrade curve of a non-degradable plas tic. The blue dot indicates when the product is discarded. The plastic starts with a performance integrity of 1, and main tains it throughout its lifespan. 3.2 Readily Degradable Plastics A readily degradable plastic is one that is able to serve its purpose effectively and then simply self-destruct at the end of its useful life. After the ti me required for useful service, during which it retains all the propert ies that it was formulated and processed to

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22 have, it simply falls apart and is assimila ted by the pervasive mi croorganisms found in nature. It returns to the ecosystem in an environmentally harmless manner. The degradation curve of an idealized, readily degradable plastic is shown in Figure 3.2. Initially the P.I is maintained at a constant level. Then, af ter a period of useful life, it is discarded and completely degrades. More over, it does so rapidly, whereupon its components are returned to the ecosystem. Some synthe tics that are vulnerable to oxidation or hydrolysis approximate such behavior. Examples include polyvinyl alcohol(PVA), polycaprolactone, and polyethylene oxide.14 Figure 3.2 Degrade curve of a readily degradable pl astic. The blue dot indicates when the product is discarded. The performance integrity starts at 1, and rapidly diminishes to almost zero when discarded. 3.3 Programmed Degradable Plastics. Programmed or controlled degradation is a new concept in which the goal is to program plastics to degrade in a predetermined time under specific conditions according

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23 to the needs of particular applications. One route to achieve degradation has been via sunlight. Photodegradation is a viable option where exposure to sunlight in most landfills is often the case. The polymer resin may be altered to make it photodegradable by attaching a photosensitizing group to the polymer chains by chemical means. When the photosensitive group is exposed to natural sun light, it absorbs radi ation, which causes the chain to break and form smaller segmen ts, a process called chain scission. Sufficient photodegradation coupled with the effects of wind and rain can eventually cause embrittlement and breakdown of the polymer. Sensitizing groups like ketones can be introduced into polymer chains of polyethylene, polypropyl ene and others, by copolymerization with an appropriate ketone monomer. Another option is to use carbon monoxide copolymerized with ethylene to produce a photodegradable polymer. The composition must be strictly controlled during the manufacture process, thereby controlling the period of time which the polymer can withstand irradiation by the sun before decomposing. Ethylene-CO compositions are mainly us ed for the large-scale manufacture of photodegradable six-pack holders for bevera ge containers. Compositions containing one percent CO will photodegrade afte r about three weeks exposure to outdoor sunlight and break up into small particles.15 As the percentage composition of the CO is increased, the weathering stability of the copolymer decreases. Concentr ations of 12% CO in the copolymer results in weathering time of the order of 40 hours or less.16 Organometallic compounds such as ferricine are known to cause accelerated oxidation when incorporated into the pol ymer blend. Salts of iron, copper, and manganese have a similar effect on polymeric degradation when the material is exposed

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24 to sunlight. A combination of photodegradati on and oxidation can be sufficient to cause appreciable degradation of dispos ed polymer, in less than 12 hours. Even with these imbedded photosensitiv e groups, there is the requirement for a certain level of stability of the product during processing, shelf-life, and sometimes, as in the case of compost bags, a period after it has outlived its desired use. It is not impossible to achieve such meticulous behavior. Metal ion complexes may act as stabilizers during processing but then decompose, after an i nduction period, in a controllable manner to form products that are photoactivators. By a careful choice of th e right combination of stabilizer and activator concentrations, the length of the induction period and the rate of the photodegradation that follows can be c ontrolled. Figure 3.3 gives a diagrammatic representation of the degrade curve of a programmed degradable plastic. Figure 3.3 Degrade curve of a programmed degradable plastic. The blue dot indicates when the product is discarded. From this time, the produc t gradually degrades and embrittles, until finally achieving a PI of zero. 3.4 BiodegradationNatures recycling.

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25 Enzymes are biological catalysts found in microorganisms, including bacteria, fungi and algae. Microorganisms contain enzymes that induce chemical degradation and are responsible for much of the chemical degradat ion that occurs in nature, and hence, the consistency of biogeochemical cycles. Microor ganisms are capable to converting organic material into carbon dioxide and methane depending on the availability of oxygen in compost sites and landfills. When sufficien t oxygen is present, aerobic degradation occurs. In the absence of sufficient oxyge n, the mechanism sh ifts to anaerobic degradation. It should be reinforced, though, that it is not uncommon to find both processes occurring within the same landfill. Mineralization is used to describe the conversion of biom ass to gases, water, salts, minerals, and residual biomass. Mineralization is complete when all biomass containing carbon is consumed, converted to carbon dioxide, and finally returned to its natural biogeochemical cycle. 3.5 Factors affecting the ra te of biodegradation. The rate at which biodegradation occurs in soil depends on soil conditions such as temperature, moisture level, degree of aeration, acidity, and the concentration of microorganisms. Under extremely unfavorab le conditions degradation rates can be reduced to nearly zero. Low temperatures severely limit degradation. This is why perishable foods are refrigerated. Moisture is also impor tant; it supports hydrolytic degradation. Newspapers, even though they are potentially biodegradable when sufficient moisture is provided, will not environmentally biodegrade in a landfill if moisture is inadequate.

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26 Finally, biotic considerations are just as important as abiotic ones. The presence of microorganisms, their quantity, whether or not they have the required enzymes for which the polymer is a substrate, the concentra tion of the enzymes, and the presence of inhibitors or predators are extr emely important. In the absence, or insufficiency of any of the aforementioned factors, degradation may be extremely slow or impossible altogether. 3.6 Degradation Considerations Degradation is defined as a deleterious ch ange in appearance, physical properties, or chemical structure of a polymer. Describing plastic degradation, sp ecifying a specific mode of degradation, measuring it and cont rolling it are all comp licated by two major factors. 3.6 (a) Plastics degrade by many routes. There are many contributors to post usag e plastic degradation before arrival, and upon residence in a landfill setti ng. Mechanical forces have the capacity to crush, tear and shred plastic into smaller pieces to onset degradation. Surface erosion processes can further fragment the polymer chains into friable powders. Wind, rain and heat all play a role in secondary degradation. Physical de terioration may also occur when water and other solvents come into contact with the plastic and leaching occurs. In general, fragments with a larger surface area to volume ratio will degrade faster than their smaller ratioed counterparts. Mechanical degrada tion is usually the prelude to abiotic degradation.

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27 Abiotic degradation is degradation in the absence of microorg anisms. This type of degradation results from reactions of the pl astic with chemicals in the surroundings like water, and acids. Alongside th e process of abiotic degrada tion, biotic degradation or biodegradation occurs. Biodegradation may e ither be enzyme catalyzed or non-enzyme catalyzed. In nature, enzyme catalyzed degrada tion is either extracellular or intracellular. Extracellular enzymes are present in the organisms environment, while intracellular enzymes are present inside the organism. Enzymes may be either endoenzymes or exoenzymes. Endoenzymes cleave internal linkages within the chain and leads to mo re rapid fragmentation, while exoenzymes cleave terminal monomer units sequentiall y. When the initial fragmentation is extracellular, polymer fragments may at some point become small enough to be transported into the cell, where degradation continues to the point of complete mineralization. Mineralization may be carried out by the same organism that provided the extracellular enzymes for the initial fragmentation or by different organisms.17 Whilst the separate effects of degradation have been compartmentalized, it is important to remember that some effects, like photodegrad ation can play a conti nuous role during the entire process. 3.6 (b) The chemical and structural composition of the plastic affects the degradation rate. Plastic degradation is heavily dependent on the chemical makeup of the plastic, including the atoms and functi onalities present on the polymer backbone. Some polymers like polyvinyl chloride, polypropylene and po lystyrene contain only carbon-carbon single bonds in their backbones. Th is feature makes them re sistant to degradation.18 A common

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28 exception to this rule is a polymer with hydroxyl groups at repeating intervals along the polymer chain. Such a polymer will be more susceptible to attack by water molecules. This behavior is referred to as hydrophilic, and is observed in polyvinyl alcohol. As a consequence of this interaction, hydrogen bonds develop between the water in the environment and the hydroxyl groups on the polymer. This promotes degradation via hydrolysis mechanisms. Polyisoprene is a natural rubber made up of a carbon atom backbone containing the double bond functionality. Such a natural rubber is environmentally degradable through the oxidation of its double bonds by atmosp heric oxygen, to produce aldehydes and carboxylic acids. The chains fragment suffici ently to promote biodegradation followed by complete mineralization. In this regard we may come to appreciate the need for antioxidants, which are usually added durin g processing to prevent excessive and undesired oxidation from occurring. Besides the nature of the chemical bonds and functionalities present, the degree of branching as well as the stereochemistry of th e molecule plays an important role in the mechanisms of biodegradation. Enzymes are norma lly specific to one particular type of chain branching and one particular stereochem istry. The percentage crystallinity as well as the extent of surface ar ea is also something worth c onsidering. In the case of polyolefins, the degree of crystallinity is im portant because oxygen does not easily enter the crystalline regions.19 Oxidation of polyolefins occu rs mainly in the amorphous regions where there is oxygen permeabilit y. A polymer with many amorphous regions formulated with antioxidant additives, would be very resilient to oxidative stress.

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29 Similarly, in polyesters, the degree of crystallinity is important because crystalline regions are less permeable to water than amorphous regions. This feature impedes hydrolytic degradation. The chemical and st ructural composition of the polymer truly illuminates the importance of stereochemistry, crystallinity and functionality as far as inherent properties relate to degradation. 3.7 The need for an alternative In keeping with the title of the thes is, the need for an alternative, biobased plastic should not only be considered in regards to the drawbacks of present, petroleum based ones, but also the benefit of moving towards a greener future. The practice of extracting energy and petrochemicals from fossil carbon is unsustainable, currently in light of the contributions to atmospheric carbon dioxide that necessarily accompany it, and over the longer term in light of the fini te nature of these resources. It is now generally recognized that atmospheric carbon dioxi de levels are being notably increased by human activity, with significant climate changes expected.20 Traditional plastics have been the subject of much debate since their popularization in the past century. Plastics have been known to lo se their additives via volatilization as in the familiar new car smell. In other a pplications, like polycarbonate baby bottles, Bisphenol A has come under fire as a contributor to developmental toxicity, carcinogenic effects, and possible neurotoxicity.21 The slow, uncalculated release of such harmful chemicals can have serious implications on mankind and other biosphere organisms.

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30 After plastics have outliv ed their useful life problems with disposal are not easy to solve. One option is to simply incinerate the used plastics and get rid of them. This however, is known especially in the case of PVC to produce toxic dioxin emissions which may build up in food chains with seri ous health consequences and environmental pollution.22 A second option is landfill disposal. Though this may be more attractive than incineration, there are still several problems with this approach. The first is obviously situational. Some countries lik e Japan do not have sufficient land to allocat e as landfill sites, which to begin with, is not usually a priority when there is greater demand for housing and other infrastructure. Even when land is allotted for such sites, there are fears that additives may leach into ground water s upplies, and cause further complications to the environment. A third option is recycling. But this re quires individual effort and calls for citizen involvement, to some extent, after these plasti cs have outlived their useful life. Collecting and sorting plastics by different types is us ually required, and in itself, is seen as a deterrent. If for example, one PVC bottle is accidentally mixed with a thousand other non-chlorinated plastic bottle s, the entire stock can suffe r from pollution stress and make recycling difficult. Furthermore, r ecycling usually produces a product of lower grade than the original, and the energy input to facilitate this conversion is sometimes impractical. In view of the aforementioned, thought should be given to th e problem that spawns such deleterious symptoms. Incineration, landf illing, and recycling are proposed solutions to the same, fundamental issue: the biodegrad able reluctance of synthetic polymers. The advantages of moving towards a greener future will be underscored in the second part of

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31 this thesis. Not only will the issue of biodegrad ability be addressed, but the sustainability of using biopolymers will be highlighted. Like most big changes in life, a paradigm shift will be necessary beforehand, to even consider the long term benefits of bioplastics in favor of synthetics. CHAPTER 4 Biopolymers 4.1 History of Biopolymers As long as there has been life, biopol ymers have been present. These are polymers that occur in nature and take many different forms, all suited to their respective functions. Biopolymers are produced by plants and animals during processes of anabolism, catabolism and other biological reactions. Of the several types of biopolymers present, the most abundant are carbohydrates and protei ns and they play important roles in nutrition. To a lesser extent are the polyesters produced by microorganisms, as well as the nucleic acids which contain genetic material. With respect to the actual man-direct ed use of these biopoly mers, it was documented that early Mayans used rubber from trees to make balls for religious games like pok-atok.23 Native Americans were also known to us e the horns and shells of bison and goats to make spoons, and other types of jewelry. H undreds of years later, the French chemist M. Lemoigne discovered polyhydroxybutyrate as a constituent of the bacterium Bacillus

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32 megaterium. Since then, PHB and related PHAs have been shown to occur in over 90 genera of bacteria.24 Biopolymers are prone to degradation, as they must be in order to take part in natures cycles. As such it would be expected that thei r chemical structure be very different from the synthetic polymers that are used in the plastics industry. Unlike the polyolefins which are resistant to degradation, the presence of nitrogen or oxygen atoms in the backbones of biopolymers make them biodegradable. It is worth exploring these biopolymers as we transition from traditional plastics to bioplastics. 4.2 Carbohydrates There is more carbohydrate on eart h than all other organic matter combined. Polysaccharides are the most abundant type of carbohydrate and make up approximately 75 % of all organic matter. There are three main types of polysaccharides that should be considered: cellulose, starch, and chitin. Of the three, the structural formulae of cellulose and starch differ only in the linkages pres ent in the glucose polymer. Cellulose is 1-4 linked, while starch is 1-4 linked. 4.2(a) Cellulose The most abundant polysaccharide is cellulose, found in plant cell walls. It forms the structural fiber of plants, keeping the cell wall in place and providi ng strength. It cannot be digested by animals, but if eaten by rumina nts it can be digested by gastric bacteria. In this way the bacteria and the animal engage in a mutualistic symbiosis. Cellulose is produced commercially from wood and cotton, a nd is used in the manufacture of paper.

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33 Modified celluloses, like cellulose acetates are used in the manufacture of paint, adhesives, ceramics and pharmaceutical film coatings. The other components of wood are lignin and hemicelluloses. Cellulose readily biodegrades via a hydrolysis reaction catalyzed by the extracellular enzyme, cellulase. Cellulose has an important place in the story of polymers because it was used to make some of the first syntheti c polymers, like cellulose nitrate, cellulose acetate and rayon. These early synthetics will be discussed in a later chapter. The structural formula of cellulose in shown in Figure 4.1 Figure 4.1Structural formula of cellulose, with 1-4 linkages. 4.2(b) Starch Starch is a very important part of the planets biomass and is metabolized to release energy for plant growth. Starch can be found in potatoes, corn, wheat, cassava and rice. The major polymer components of starch ar e amylopectin and amylase. The relative compositions vary depending on the type of pl ant. Corn starch is roughly one quarter amylase and three quarters amylopectin. The ma jority of it is used to make sweeteners and as chemical feedstocks in various i ndustries. It is used for example during fermentation to make polylactic acid, a biopol ymer. The structural formula of starch is shown in Figure 4.2.

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34 Figure 4.2Structural formula of starch, with 1-4 linkages. 4.2(c) Chitin Chitin is another abundant polysaccharide and it is found in the skeletal systems of marine crustaceans. Chitin is produced mainly from shellfish waste and constitutes one part of the hard composite material formi ng the exoskeleton. An important derivative, chitosan, is produced commercially from chitin by a base-catalyzed deacetylation reaction. Both chitin and chitosan have appl ications in cosmetics, personal hygiene products, agriculture, and food ingredients. Th ere is strong interest in finding additional applications for this abundant polymer. 4.3 Proteins Proteins are polymers formed by the condensation polymerization of amino acids. The carboxyl group of one amino acid joins w ith the amino group of another amino acid, eliminating a water molecule and forming a pe ptide bond (a type of amide linkage). Each specific protein is made up of a characterist ic sequence of amino acids. The structural formula of a protein in terms of its amino aci d residues is shown below, in which R varies from residue to residue and identifies the amino acid from which each residue is obtained. If the complete protein sequence is known, then the chemical formula may be expressed in terms of the ordinal naming of each residue. The structural formula for a protein is shown in Figure 4.3.

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35 Figure 4.3 Structural formula for a protei n, where R varies from residue to residue. There are twenty amino acids that are co mmon to all living organisms. Some proteins have a structural function, like collagen and keratin while others are enzymes. Among the abundant animal proteins are collage n, casein, whey protein and keratin. 4.3(a) Collagen Collagen is an abundant mammalian protein found in tissues and internal organs. It is insoluble and fibrous and constitute s thirty percent of total protein. 4.3(b) Casein Casein is a phosphoprotein found in milk. It has relatively little secondary structure or tertiary structure and, because of this, it cannot be denature d. It is relatively hydrophobic and is found in milk as a suspension of particles called casein micelles. It is used as a labeling adhesive in the bottling industrie s on account of its excellent rheological properties and in binders and protective coatings.25 4.3(c) Whey Whey is the soluble fraction of milk that is separated from the casein curd during cheese manufacture. Whey protein, isolat ed from whey, has several important

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36 applications in the food industry, and bulki ng agents for body building. It is easily absorbed, and readily bioa vailable, once in the body. 4.3(d) Plant Protein Plant proteins are obtained from soy, zein (from corn), potato, peas, and other legumes. Soy protein is used for making co atings for paper and paperboard. It was originally used by Henry Ford for his early bi oplastic initiatives. Zein is used in grease proof paper coatings since it has goo d barrier properties to oils. 4.3(e) Amino Acids Amino acids can be polymerized. One example is poly-(aspartic acid) (PAA), which has been used as a biodegradable substitute for synthetic polyacrylate. PAA has also been used as a mineral scale inhibitor in water tr eatment applications, a dispersing agent in detergents to prevent the deposition of soil on surfaces, and a dispersing agent for pigments in paints. 4.4 Polyesters Polyhydroxyalkanoates are natura lly derived polyesters made from the polymerization reactions of hydroxyalkanoa te monomers. These monomeric building blocks are produced by bacter ia, and occur as inclusion bodi es deposited as granules inside the cytoplasm. Whenever carbon is in excess, but some other nutrient limits growth, PHA granules accumulate and serv e as energy and carbon storage materials, being consumed when no other carbon source is available. The most common PHA in

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37 nature and the first to be discovered wa s poly-3-hydroxybutyrate (P3HB). Since then others have been discovered, in cluding poly-3-hydroxyvalerate(P3HV).26 Naturally occurring polyesters, like the polyhydroxyalkanoa tes are now produced commercially from microorganisms through b acterial fermentation. In bioreactors, microorganisms are fed a carbon source substrate that serves as a means of preparation for a specifically desired polymer. For the pr oduction of PHB, glucose is used as the substrate in excess, while for PHV, the de sired feedstock is pr opanoic acid. Propanoic acid is obtained from the fermentation of wood pulp waste or from petroleum. Polyhydroxyalkanoates are produced in a two-stage fermentation process. The first consists of cell growth followed by polymer accumulation.27 This can proceed to as much as eighty to ninety percent of the cells dry weight. To harvest the polymer, the cell wall is ruptured and the polymer is collected and purified. For m=1 R= CH3 3-hydroxybutyrate 3HB R= C2H5 3-hydroxyvalerate 3HV R= C3H7 3-hydroxycaproate 3HC R= C4H9 3-hydroxyheptanoate 3HH R= H=1 m=3 4-hydroxybutyrate 4HB m=4 5-hydroxyvalerate 5HV Figure 4.4Chemical structure of major PHAs. Production of polyhydroxyalkanoates has r eached approximately 1 million pounds a year and is increasing with renewed interest and vigor from bioengineering companies. They are biocompatible as well as biodegradab le and have biomedical applications in the

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38 areas of controlled drug release, surgical su tures and others. The structural formula of a PHA is shown in Figure 4.4. As R, and m va ry, different types of PHAs are produced. 4.5 Natures fibers This section will briefly introduce the comp lex, yet highly organi zed structural units of natural fibers like cotton, wool and sil k. Natures way of combining strength and functionality is impressive, and in some wa ys, mankinds most astonishing infrastructural marvels, model nature very closely. 4.5(a) Cotton and Wool Natures use of biopolymers to produce stro ng materials is indexed by the presence of fibers such as cotton, wool and silk. These fibe rs have been used in the clothing industry for many years because of their strength. Cotton is a plant fiber made up of approximately 90% cellulose. Impurities, such as seeds, are removed and the cotton is then bleached and sterilized. Wool is the sheared hair from sheep. The main constituent of wool is called fleece, and it is quite popular for making sweaters and mittens. Lanolin (a component of animal grease), is sometimes left as a blend within the fleece on account of its water resistant properties. Sweaters such as these are partic ularly useful where warmth and dryness are simultaneously desired. Fishermen on whalers, in colder climates make good use of this property. 4.5(b) Silk

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39 Silks are natural protein fibers that are externally spun by the silkworm moth caterpillar, although a similar type is produced by spiders. Si lks combination of strength and flexibility is the direct result of the polym ers chemical structure. Within the fibroin molecule there are regions of approximately sixty amino acid residues consisting of a repeated hexapeptide sequence. These sixty residue regions form ordered helical chain conformations, but are separated within the ch ain by more flexible units of around thirty amino acid residues. The ordered segments p ack together in bundles to give silk its strength; the amorphous connecting segmen ts provide reduced crystallinity and flexibility.28 The shimmering appearance for which silk is prized comes from the fibres' triangular prism -like structure which allows silk cloth to refract incoming light at different angles. Natural fibers can be very strong. Silkworm s ilk is as strong as nylon; spider silk is stronger than nylon. On a strength to weight basis both outperform steel. Natural fibers illustrate the high performance character of natural biodegradable materials. But even more impressive is the way living systems ha ve evolved to create high-performance and programmed degradable composites.29 4.6 Natures composites A composite is a solid product consisting of two or more distinct phases. As a result of biological evolution, nature has produced extraordinary materials serving complex and diverse functions. It is worth considering co mposites based on three different parameters and their respective combinations. These parame ters are strength, elasticity, and hardness. In blood vessels and skin, strength is combined with elasticity. In bones, strength is

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40 combined with less elasticity, but with the ability to flex under stress. In teeth, strength is combined with extreme hardness. Nature uses a hierarchical system of structure combined with the use of composites. In skin, for example, a low level of cross-linking allows for flexibility and softness. The cross linked keratin fibrils are embedded in a matrix of other proteins to produce a fiber-reinforced composite. One such protein is elastin, which, as its name suggests, is mainly responsible for the el astic properties of tissues such as skin, blood vessels, ligaments and lungs. Natures design for plants is also impressive. Wood, as a material, has to be strong enough to support the numerous gr een leaves and fru its on trees. But it must also provide circulatory systems for the transport of water, nutrients and sugars provided by photosynthesis. Strength is provided by cellulose fiber cell walls. Within the cell walls the cellulose fibers are laid out in crissc ross reinforcing patterns and are embedded in a matrix of other molecules, mainly lignin. A lignin/cellulose composite may be likened to reinforced concrete, where cellulose serves a purpose similar to reinforcement bars, while the lignin serves the function of concrete. In the space between ce lls, are other organic molecules, including hemicelluloses and more lignin.30 Natures composites provide many desirable properties like strength, elasticity and hardness. Equally as important, is the fa ct that these compos ites biodegrade in a programmed manner. Very rapid environmenta l biodegradation is us ually not the norm. By modeling natural biodegrad ation cycles it is possible to create biopolymers with similar degradation patterns that increase func tionality and make dis posal concerns trite, allowing nature to work on our behalf.

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41 CHAPTER 5 Bioplastics 5.1 Introduction to Bioplastics Bioplastics are biodegradable polymers whose components are derived entirely or almost entirely from renewable raw materi als or microorganisms. Biopolymers possess a variety of chemical structures and are nont oxic and biodegradable. Such properties make biopolymers a potential source of feedstock for biodegradable plastics. During the commercial processes of extraction and purification, the native plasticizing agents in the bioplastics may be removed, but plasticizing agents can be reintroduced to produce non-brittle bioplastics. Other additives can enhance their properties. The final formulation may consist of one or more biopolymers combined with one or more plasticizing agents, and one or more additives. Each component will contribute particular proper ties to the final physical and chemical behavior. Some biopolymers are thermoplastic and can be processed with the same methods used with synthetic polymers, such as extr usion or compression molding. An example of

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42 this is thermoplastic starch. With biopolymers that are not thermoplastic, sheets can be made by casting. Casting is a process that invol ves continuous solven t removal, initially producing a gel state, and eventually a solid bioplastic material. Because many biopolymers are soluble in water, bioplastic technologies ar e often water-based. There are some low-molecular weight biomolecules that may be polymerized to form thermoplastic materials, and others that might involve cros s linking to produce strong thermosets in the final stages of polymerization. Unprocessed biopolymers are essentially bi odegradable, as they must be to take part in natures cycles. For processed biopolymers to be biodegradable in a specific environment, these biopolymers must be a pot ential substrate for the enzymes present in this environment. Specific microorganisms must be present and in sufficient quantity for effective biodegradation to occur. In some instances, when biopolymers are chemically processed, the processed materials may or ma y not be a substrate for the same enzyme that degrades the biopolymer. In such a cas e, compartmentalized degradation may occur where the biopolymeric material may be subjec t to degradation while other parts may not. A closer look should be taken at the early bi oplastics as a transition is made to the new bioplastics. 5.2 Early Bioplastics The innovativeness of mans early use of biopl astics is discussed in this section on the early bioplastic s. These biopolymers spanned hundreds of years of use, as cutlery articles, paint dyes, jewelry, automobile parts, and billiard balls. 5.2 (a)

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43 Keratin and Gelatin Bioplastics have been around for qui te some time. Animal parts, including mammalian bones and horns contained keratin which was boiled and shaped to make spoons and ornaments. Gelatin was produced when collagen was boiled in water. When cooled, the solution formed a gel with a mol ecular weight one-third that of collagen. From this observation, treatment of boiling water somehow separated the strands of the helix in collagen, breaking the inter-chain hydrogen bonds, and replacing them with hydrogen bonds to water molecules.31 When gelatin was mixed at high concentrations with water and plasticizer, it formed a slurry or viscose. When dried, the viscose formed a tough, hard solid. Bottle caps and printer rollers were made from gelatin on a large scale.32 5.2 (b) Casein Casein obtained from milk has been used in the production of plastics since about 1919.33 A synthetic wool-like fiber known as Lanital was made from casein during the 1930s. Such synthetic fibers were good subs titutes for wool and aided conservation efforts during WWII. At the end of the ei ghteenth century, Adolf Spitteler invented moldable, casein plastic by mixing form aldehyde and milk. He called it Galalith.34 The commercialization of Galalith involved isolating pure casein from buttermilk, mixing it with water, and extruding the mixture into a formaldehyde bath. In the formaldehyde bath, cross linking produced a so lid, which hardened and cured.35 5.2 (c) Soybeans

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44 Henry Ford, the automobile magnate, at the turn of the 20th century was motivated by a desire to find nonfood applica tions for agricultural surpluse s. It seemed feasible to channel his efforts towards his business, and he sought means of using soybeans in the production of automobile paints, rubber substitutes, upholstery cloth and other applications. The composition of soy plasti cs varied according to their intended application in the automobile. The core resin involved mixing soy meal with formaldehyde to produce a strong, cross-linked protein. In cases where added strength was desirable, the protein matrix was co-condensed with phenol or urea. During cocondensation, cellulose-based fillers were added from wood and co tton. The final product contained as much as 70% cellulose and twen ty percent soy meal. If further strength was desired, glass fiber was also used in the formulations.36 5.2 (d) Ebonite and Gutta Percha In the nineteenth century, one of th e first bioplastics to be commercialized was ebonite, a black vulcanized form of rubber. It was processed into combs, brushes and electrical insulation. It was the brainchild of Mr. Charles Goodyear. Gutta percha, Figure 5.1, a natural latex, was obtained from the sa p of tropical trees hundreds of years ago. Synthetic gutta percha is polymer ized from isoprene and has the trans configuration. It has several applications on account of its bioinertness, resilien ce and high dielectric strengths.37 Some applications include underwater cable insulation, golf balls, ornamental frames, and other objects.

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45 Figure 5.1 Trans-configurational polymer of gutta percha. 5.2 (e) Cellulose Nitrate Cellulose nitrate, Figure 5.2, is obtaine d by the action of a mixture of concentrated nitric and sulfuric acids on cellulose at a temperature below 400 celcius. It was first prepared in 1845 when C. Friedrich discovere d a paper derivative that was strong and water resistant, yet very explosive. The next year, L. Menard produced a solution of cellulose nitrate in ether-eth anol and called it co llodion. It could be dried into a tough, elastic and water proof solid. During the next five years, applications of collodion in the fields of photographic duplication as well as medicine were pursued with interest. Figure 5.2 Cellulose nitrate polymer. In 1862, A. Parkes pressure molded collodion to create Parkesine. He began to publicly market his product by producing comb s, knife handles, buttons and other items. A few years later, he discovered that by adding 2-20% camphor during processing, parkesine could achieve better texture uniform ity and contractile properties. Again, he decided to commercialize the new product, and soon umbrella handles, chess pieces and

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46 jewelry were being marketed. Unfortunately, af ter two years, his company failed due to a host of reasons ranging from inferior ra w materials, over-rapid production, and the flammability of parkesine.38 By the middle of the 19th century, global se nsitization to the endangerment of elephants demanded a substitute for ivory. At the time, the principle application of ivory was in billiard balls on account of its good rebounding properties. Ma ny substitutes were considered, but most failed on account of poor physical properties. Eventually, in 1869, J. Hyatt piggybacking on Parkes discovery of parkesine, decided to use camphor-ethanol as a plasticizer, instead of a solution, for he lping liquid collodion dry to a useful material. Hyatts composition was called celluloid, and it made the solid moldable: a thermoplastic.39 5.2 (f) Cellulose Acetate Although there were many advantages of cellulose nitrate, there was one drawback that challenged chemists to find an alternative for several of its applications. This drawback was its flammability. Cellulose acetate, Figure 5.3, proved to be the answer. Cellulose acetate is produced when cotton fiber reacts with acetic acid, and acetic anhydride using an acid catalyst. Its deve lopment was stimulated by the 1914-18 war where it was used as a fire-proof dope fo r treating aircraft wings and fuselages.40 Cellulose acetate is thermoplastic and was processed by many of the same methods as cellulose nitrate, includi ng extrusion and casting.

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47 Figure 5.3 Cellulose acetate polymer. 5.2 (g) Rayon and Cellophane Alcohols, when treated with carbon disulphide and aqueous sodium hydroxide, form compounds called xanthates. Cellulose is cap able of undergoing a similar reaction to form cellulose xanthate.41 Cellulose is composed of both crystalline and non-crystalline regions. In an aqueous envir onment, the crystalline regions are impermeable to water, while the amorphous, noncrystalline regions, swell.42 In the presence of alkali, cellulose xanthate dissolves to form a viscose. When this viscose is forced through a spinnerette into an acid bath, cellulose is regenerated in the form of fine filaments which yields threads of the materi al known as rayon. If viscose is forced through a thin slit, cellulose is regenerated as thin sheets which, when softened by glycerol, are used for protective films. The end product is called cellophane. Even though rayon a nd cellophane are referred to as regenerated cellulose, it should be noted that alkali degradation cause s the final product to have shorter chains, and less crystallinity than the original cellulose.43 The product therefore interacts much more strongly with water and although it ha s high water-vapor permeability and loses strength when wet, it does have goo d resistance to oils and greases.

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48 With the emergence of the petroleum industry in the 1900s, much was done in terms of shifting from bioplastics to synthetics. Pe troleum was seen as an abundant source of chemical feedstock as well as a cheap and accessible form of fuel. Phenolics were commercialized in 1907 and bakelite provided nonivory billiard balls, replacing celluloid. Many other plastics followed in the 1920s. World War II brought on a large increase in plastics production, a growth that continues to this day.44 5.3 The New Bioplastics With fossil reserves depleting and green thinking emerging in todays society, there has been rejuvenated efforts in the field of bioplastics. Companies like Metabolix, based in Cambridge, Massachusetts have been ackno wledged for their efforts in the field of biodegradable plastics and have recei ved numerous awards including the 2005 Presidential Green Chemistry Challenge award. According to Dr. Oliver Peoples This award recognizes Metabolixs success in tr ansforming PHA Natural Plastics technology from a biological curiosity to a commercial reality.45 With this in mind, let us consider the following three methods of commercially obtaining biopolymers. 5.4 Biopolymers extracted directly from their natural origin. Biopolymers may be extracted from their na tural origin in the form of carbohydrates, proteins. These biopolymers may be used separately or in conjunction w ith synthetic derivatives depending on the re quired application and pref erred degradation rate. By meticulously varying polymeric compositions, a range of synergistic susceptibilities may

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49 be characteristic of a specific polymer. For instance, solubility, may onset fragmentation, which will encourage biodegradability. 5.4 (a) Carbohydrates-Starch Starch is one of the mo re popular examples of a biopolym er extracted from its natural origin. It is abundant, cheap, and becomes th ermoplastic when properly plasticized with water. This formulation is known as therm oplastic starch (TPS). TPS has an amylose content greater than 70% and is based on gela tinized vegetable starch. With the use of specific plasticizing solvents, it can pr oduce thermoplastic materials with good performance properties and inherent biodegrad ability. Starch is typically plasticized, destructured and/or blended with other materi als to form products with useful mechanical properties. Biodegradation of st arch based polymers is a result of enzymatic attack at the glucosidic linkages between the sugar groups, leading to a reduction in chain length and the splitting off of sugar units that are readily utilized in biochemical pathways. 46 Polyvinyl alcohol is a synthetic, hydrophilic polymer on account of the (OH) functionality on successive carbon atoms. It can be blended with starch to produce thermoplastics which can be processed by extrusion and other molding techniques. Products can be made with varying degrees of solubility which depend on presence, and amount, of the amount of polyvinyl alcohol co mponent as well as its crystallinity. Crystallinity of PVA is heavily dependent on the amount of residua l acetate left on it, after partial or complete hydrolysis of polyvinyl acetate. Useful applications include products that could be flushed after use, which could easily disso lve and subsequently biodegrade in sewerage sites.

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50 Another application of biodegradable starch is in the packaging industry. Starch foams may be extruded to form peanuts wh ich are functionally si milar to polystyrene, but are biodegradable and water soluble. Ble nding starch with polyvinyl alcohol increases the water resistance of the foam (as acetat e component increases, water solubility decreases) and articles like cups and plates can be shaped and produced. Starch foam can also be made more water resistan t without using a synthetic polymer, by derivatization. Derivatization is a chemical modification whereby some of the hydroxyl groups (OH) are converted into acetyl groups (-OCOCH3). The acetylated starch foam has a higher water resistance but remains biodegrad able. Alternatively, starch foam can be coated with a layer of acetylat ed starch, but the best result s are obtained when acetylated starch is used to coat a st arch-acetylated starch blend.47 5.4 (b) Chitin and Agar There are other abundant polysaccharides lik e chitin and agar which can be directly extracted from their natural source. Agar for example, is obtained from the cell walls of seaweed and has many applications in mo lecular biology. Chitin is found in the exoskeleton of crustaceans and insects. Chitin is not thermoplastic, but can be prepared as low-oxygen permeability films by evaporation of so lvent. Paper towels that claim to have high wet-strength are usually com posed of a chitin/cellulose blend. 5.4 (c) ProteinsSoy, Zein and Gelatin.

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51 As mentioned in the section on Ea rly Bioplastics, soy protein obtained from soybeans, can be processed using extrusion and injection molding techniques to form combs, personal articles, credit cards and others. Zein is thermoplastic and water insoluble. It resists microbial attack and forms fibers that are strong, washable, dyeable, and grease resistant. Gelatin is widely us ed for drug and vitamin encapsulation. When plasticized properly, these prot eins form flexible films which may be applied to preserve flavor, maintain optimum moisture content, enhance freshness and pr ovide protection, for consumable products.44 Polysaccharides and proteins are already in large-scale commercial production. They are available for use as bioplastics feedstocks. Starch is inexpensive and easily accessible. It is also thermoplastic. Its limited physical properties ca n be easily ameliorated through chemical modifications, coatings, and in comb ination with other biopolymers in blends and composites. 5.5 Biopolymers produced from fermentation (Polyesters) Fermentation is a process whereby micr oorganisms are subjected to conditions that stimulate anaerobic respirati on. Today, there are two ways in which fermentation may lead to the production of biopolymers. B acterial fermentation, and Lactic Acid fermentation are discussed in the next two sections. 5.5 (a) Polyesters produced from bacteria Bacteria such as R. eutropa or E. coli are capable of producing polyesters known as polyhydroxyalkanoates (PHAs). Common PH As include polyhydroxybutyrate and

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52 polyhydroxyvalerate (PHV). PHB has limited applications though, because of its brittleness. On the other hand, a copolymer of P3HB and P3HV, PHBV, can display a wide range of properties depending on the ratio of each monomer in the copolymer. PHBV is produced when propanoic acid is added to glucose during fermentation. The ratio of glucose to propanoic acid determines the composition of the copolymer and this determines its physical properties. P3HB accumulates as discreet granules in side the bacteria, to approximately 70-80% dry weight. At the end of fe rmentation, cells are disrupted by heat and the polymer is purified. Purification in volves using enzymes and detergen t washes to solubilize cell components and purify the water insoluble polymer.48 A white powder is produced which is thermoplastic. It is water resistant and oxygen impermeable and hence, suitable for a wide range of applications lik e bottles and films. PHBV is also biodegradable in marine and freshwater environments, compost sites an d sewer systems and is easily mineralized to carbon dioxide and methane by microorga nisms to the point of eighty percent molecular weight in one month. Its biodegradation rate, lik e its physical properties, depends on copolymer composition, molecula r weight, degree of crystallinity, surface area, and the presence of biodegradable additives such as plasticizers. Biodegradation begins with bacteria or fungi colonizing the surface and excreting an extracellular depolymerase enzyme that degr ades and solubilizes the polymer near the cell. Fragments are then abso rbed through the cell wall and mineralized. Moreover, the formulation of PHBV makes it compatible with recycling and clean incineration.49 Table 5.1 lists polymers that can be obtained directly from their natural environment. Biopolymer Natural Source What is it?

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53 Cellulose Wood, cotton, corn, wheat, and others This polymer is made up of glucose. It is the main component of plant cell walls. Soy protein Soybeans Protein which naturally occurs in the soy plant. Starch Corn, potatoes, wheat, tapioca, and others This polymer is one way carbohydrates are stored in plant tissue. It is a polymer made up of glucose. It is not found in animal tissues. Polyesters Bacteria These polyesters are created through naturally occurring chemical reactions that are carried out by certain types of bacteria. Table 5.1 Biopolymers extracted directly from their natural environment. Polyesters are produced from controlled fermentation processes. 5.5 (b) Lactic Acid Fermentation In the strictest sense, polylactic acid (PLA) is a biopolymer, but not one that is obtained directly from living organisms. Lac tic acid is produced from the fermentation of glucose, of which common sources include su gar beets and sugar cane, as well as the products of starch conversion from corn or potato. There are us ually high yields of lactic acid (> 90%) which help to make fermentation economically favorable. One way of polymerizing lactic acid i nvolves using metal-catalyzed, ring-opening polymerization of the cyclic dimer of lac tic acid, to produce a high-molecular weight polymer. Another common method for PLA synt hesis is via condensation polymerization of lactic acid. Compared to the ring-ope ning method, polymerization via this route produces a low molecular weight polymer. PLA-based polymers are not water soluble, but microbes in marine environments can degrade it into water and carbon dioxide.50 In addition to PLA, triglycerides represent anothe r form of polymer that may be indirectly obtained from plants, and seeds.

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54 Figure 5.4polymerization of lactic acid to form polylactic acid (PLA) using dimeric ring-opening. 5.6 Triglycerides Triglycerides represent the chemical forms in which fats and oils are stored in plant and animal cells. Triglycerides have become the basis for a new family of sturdy, durable composites that have long, useful life-times. See figure 5.5. Figure 5.5Structural fo rmula of triglyceride. The technique of epoxidation is used to c onvert unsaturated trig lycerides to a more chemically reactive form capable of polym erizing. The initial li quid resin is a lowmolecular-weight polymer that is then co mbined with catalysts and accelerators to facilitate a cross-linking reaction. The resin is injected into a mold containing a reinforcing fiber, and then heat-cured to form a rigid thermoset.51 Biopolymer Natural Source What is it? Lactic Acid Beets, corn, potatoes, and others Produced through fermentation of sugar feedstocks such as beets, and by converting starch in corn, potatoes, or other starch sources. It is polymerized to

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55 produce polylactic acid -a polymer that is used to produce plastic. Triglycerides Vegetable oils These form a large part of the storage lipids found in plant and animal cells. Vegetable oils are one possible source of triglycerides that can be polymerized into plastics. Table 5.2Biopolymers obtained indirectly from natural sources which are polymerized to produce bioplastics. 5.7 Biopolymers produced from plants Plants are capable of producing the same polyesters as in bact erial fermentation. The technique of using plants as biofactories is the newest advancement in PHA production. Microbiologists have been able to express the A. eutropus genes encoding the acetoacetyl-CoA reductase (phaB) and PHA synt hase (phaC) in plants under the control of CaMV 35S promoter.52 This novel pathway leads to reasonably large quantities of PHAs instead of the endogenous pathway that leads to lipid formation. It is envisaged that plants like Arabidopsis thaliana tobacco and Panicum virgatum could be capable of producing bioplastics as part of regular cellula r processes. See Discussion, chapter seven, page 84. 5.8 Biodegradable synthetic polymers All biopolymers are biodegradable, but not all biodegradable polymers are biopolymers. Some synthetics, merely by virtue of the presence of ce rtain functionalities in their polymeric backbone may be susceptibl e to various methods of degradation, or at least some manner of fragmentation. There ar e two main mechanisms of degradation, either following a pathway of oxidation or a pathway of hydrolysis.

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56 Hydrolysis is a chemical reaction that involves using water to break down a polymer. It is technically the opposite of condensat ion, and so, by absorbing water, in suitable conditions, the polymer may re-monomerize. Oxidation is a chemical reaction that involves the addition of oxygen to a molecule or the removal of hydrogen. From this knowledge, consider the struct ural formulae of the followi ng polymers in Figure 5.4. Note that polyglycolic acid (PGA), as well as polycapro lactone (PCL) possess ester linkages and can readily degrade via hydrolysis to form th e corresponding monomer or educt. Figure 5.6 Some biodegradable synthetic polymers. Po lyglycolic acid is used extensively in the medical field. CHAPTER 6 Medical Applications of Polyhydroxyalkanoates. So far, we have seen the commercial benefits of PHAs and their plethora of uses. Bioplastics have proved themselves to be fu nctionally equivalent to plastics in all spheres, from production to usage. But there is one more advantageous benefit of making Polyvinyl alcohol Polyethylene oxide Polycaprolactone Polyglycolic acid

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57 a transition to bioplastics. If nothing else was to be gained, this one application would be worthwhile in itself. Poly-4hydroxy butyrate (P4HB) is a polyester that is currently being developed as a new absorbable bi omaterial for medical applications. The biomaterial offers a new set of propertie s that significantly extends the existing biomaterial design space allowing the de velopment of new and improved products. In the field of cardiovascular research, for example, the use of P4HB has resulted in the first successful demonstration of a tissue generated tri-leaflet heart valve in a sheep model. Other products under development incl ude vascular grafts, stents, patches, and sutures.53 In order to appreciate the use of PHAs in the medical field, their production, properties, processing techniques, sterilizatio n practices and in vivo biocompatibility will be addressed. Wherever necessar y, in keeping with the idea of a better alternative, PHAs will be compared to other synthetic polymers in the medical field. 6.1 Production Polyhydroxyalkanoates are a class of polyesters produced by certain microorganisms and stored as minute granules inside their ce lls. These intracellular granules function as energy and carbon storage. PHAs are produced via fermentation, using sugars and oils together with other co-feeds. When the granules accumulate to 90% of the cells dry mass, they can be isolated by breaking open the cells and using either an aqueous-based or solvent-based extraction process to rem ove cell debris, lipids, nucleic acids, and proteins. Initially, certain mi croorganisms were capable of producing these polyesters. By the late 1980s the genes responsib le for PHA production were isolated54 and transgenic procedures were developed fo r greater large scale production using genetically modified

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58 Escherichia Coli K12 microorganisms. It was observe d that the structure of P4HB closely resembled that of other chemica lly derived polyesters and possessed similar properties. 6.2 Mechanical Properties The mechanical properties of P4HB can be varied and they span a fair range in terms of strength, flexibility and hardness. Poly merization of 4HB with other hydroxy acids such as 3-hydroxybutyrate (3HB) for example, can yield elastomeric compositions at moderate 4HB contents (20-35%), and rela tively hard, rigid polye sters at lower 4HB content (less than 20%). The flexibility of a polymer is a measure of its extension before breaking, while its toughness gi ves an indication of impact strength. Tensile strength is the minimum force required to cause breakin g in a polymer specimen. P3HB is a relatively stiff, rigid material that has a tensile strength comparable with that of polypropylene, while P4HB has a tensile stre ngth that tends to ultrahigh molecular weight polyethylene. As a homopolymer, P4HB possesse s extremely high tensile strengths, flexibility and has an extension to break of around 1000%. This is two orders of magnitude greater than P3HB. There are several ways of altering th e mechanical properties of P3HB. One interesting find was that extending the dist ance between the ester groups in the PHA backbone can have a dramatic impact on m echanical properties. Also, combining different monomers to form copolymers, as in poly(3HB-co-4HB), pr oduces one series of materials with a wide range of useful mech anical properties that can be tailored to specific needs.55

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59 The properties are compared in table 6.1, show ing individual propert ies of 3HB and 4HB, alongside properties of one of their copolymers. PHA Poly(3HB) Poly(4HB) Poly(3HB-co-16%4HB) Melting Temperature/ C 177 60 152 Tensile Strength/ MPa 40 104 26 *Tensile Modulus/ GPa 3.5 0.149 Not determined Elongation at break/ % 6 1000 444 Table 6.1 Comparison of P3HB P4HB and their co-polymers. *Tensile modulus is the ratio of stress to elastic strain in tension 6.3 Processing The processing techniques for P4HB depend heavily on its molecular weight. High molecular weight P4HB requires temperatures in excess of 800K du e to a high melt viscosity. On the other hand, lower molecular weight P4HB can be melt processed fairly easily. P4HB is also soluble in polar solvents and is particularly useful in solution coating, phase separation techniques to make porous devices, and preparation of microspheres, and could facilitate solutionspinning methods. P4HB also appears to be much less sensitive to hydrolysis by atmosphe ric or residual moisture than synthetic absorbable polyesters derived from -hydroxy acids. While minimizing moisture will be important during processing of P4HB, rigorous drying or exclusion of moisture may not be necessary.56 6.4 Sterilization

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60 Sterilization of PHAs usually involve treatment with ethylene oxide, and has not been observed to cause any changes or implications to the physical or chemical properties of the polymer. However, PHAs that have been fa bricated ready for use are sterilized using a cold cycle due to the low melting point of the biopolymer. A second method of sterilization involves the use of gamma ra diation. In comparison to ethylene oxide, however, there is an apparent reduction in mo lecular weight of the polymer as well as increased polydispersity (the state of nonuniformity in molecular weight) of the sample.57 6.5 Biocompatibility As far as the in vivo consideration of PHAs, P4HB is not only biocompatible, but it is well tolerated biologically. In fact, the hydrol ysis of P4HB yields 4-hydroxybutyrate, a natural human metabolite presen t in the brain, muscle and liv er. 4HB has a half life of about thirty minutes and is eliminated from the body via the Krebs cycle in the form of carbon dioxide. Besides this, concerns over the retention of enhanced amounts of 4HB in the body, stem from its use as an intravenous agent for the induction of anesthesia and a treatment for narcolepsy. However, since the ha lf-life of the acid is relatively short, and multi-gram doses are required to obtain any hypnotic effect, small implants of P4HB could not induce general sedation, for example.58 Apart from 4HB, low molecular-weight forms of P3HB have also been detected in human tissues, in blood serum complexed w ith low-density lipoproteins and in human aorta. 6.6 Absorption

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61 P4HB has been observed to degrade at different rates relative to other synthetic polyesters as well as other biopolymers. It de grades slower than polyglycolic acid (PGA), but faster than other polyhydroxyalkanoates lik e P3HB in a subcutaneous environment. In one implantation study, it has been repor ted that the loss of mass from a P4HB implant varies with porosity. When solid 50, and 80% porous samples were implanted subcutaneously in rats, the average mol ecular weight of the polymer decreased significantly but independently of sample conf iguration. However, these samples lost 20, 50 and almost 100% of their mass, respectiv ely, over a ten week period, suggesting that degradation of P4HB in vivo depends in part on surface area.71 Furthermore it follows that implants of P4HB are likely to undergo gradual changes in mechanical properties rather than the more abrupt changes observed with other synthetic absorbable polymers, like PGA. T his could potentially be advantageous in applications where a sudden loss of a mechani cal property is not desirable, and a steadier loss of implant mass with concomitant growth of new tissue is beneficial.59 Finally, the degradation rate of PHAs into acidic products is not as rapid as other synthetics like PGA. This is another benef it of using PHAs instead of -hydroxy polyesters, and ensures that internal PHs of tissue fluid and surrounding membranes are not significantly affected. 6.7 Applications This section will underscore the importance of the PHAs in applications such as congenital cardiovascular defects, heart valv es, sutures, grafts and bulking agents. The

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62 more popular PGA will be contrasted alongsid e P4HB as we explore one of the most fascinating applicati ons of bioplastics. 6.7 (a) Congenital cardiovascular de fects artery augmentation. According to the American Heart Association, every year, around forty thousand babies are born in the United States with c ongenital cardiovascular defects. They are the most fatal kinds of birth defects. Traditional means of dealing with this problem has been to use synthetics like polytetrafluoroeth ylene (PTFE) patches. However, a better alternative would be to use living tissue as a scaffold to promote healing. With this in mind, much research has been done, incorporating P4HB in tissue scaffolds. Patches of P4HB with porosities in exces s of 95% and pore si zes in the range 180240pm were prepared through a combination of salt-leaching and solvent evaporation. These scaffolds were seeded with autologous endothelial, smooth muscle, and fibroblast cells prior to being implanted to augment the pulmonary artery in a sheep model. Six cellseeded patches and one unseeded control were used in the 24-week study. At 4, 7, and 24 weeks, echocardiography and examination of the cell-seeded explants revealed progressive tissue regeneration with no evidence of thrombus, stenosis or dilation. In comparison, a slight bulging and somewhat less tissue regenerati on were noted at 20 weeks for the control patch.60 The results demonstrate the feasibility of developing a P4HB tissue scaffold for use as a cardiovascular patching material. Another interesting feature of P4HB is its ease of surgical use compared with PTFE. This ease of use directly relates to the high elongation capacity of P4HB, a crucial property for vascular grafts. In addition, upon implantation of P4HB patches, no bleeding

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63 is observed along the suture line, in comparis on to the PTFE patches. Finally, in the PTFE patch, the suture leaves a hole for blood to leak thro ugh, while the P4HB patch is self-healing, thus preventi ng blood leakage. Table 6.2 belo w compares P4HB and PGA. Property PGA P4HB Thermal properties High melting temperature Low melting temperature Tensile strength Very strong Strong Tensile modulus Stiff Flexible Ability to elongate Virtually none High Absorption rate Very fast Moderate Loss of strength in vivo Rapid Gradual Loss Degradation products Highly acidic Less Acidic Inflammatory reaction Severe for large implants Well tolerated Thermoplastic melt processing Yes Yes Solvent processing Virtually insoluble Soluble in range of solvents Resistance to moisture Poor Fairly good Table 6.2 -Comparison of physical and chemical properties of P4HB and PGA. 6.7 (b) Heart valves Some of the most astounding results in heart valve developments have been obtained using P4HB. Heart valve surgery is a common practice in the United States especially with infants. Existing heart valve prosthes es are non viable and young patients often outgrow replacement valves and need repeat surgeries to replace them. Furthermore,

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64 improvements are also needed for the adult population to improve valve durability and reduce the need for anticoagulant therapy. A tissue engineered heart valve may prove viable in addressing these issues. In an early study to create a tissue e ngineered heart valve, synthetic absorbable polyesters were used as scaffolding material for heart valves. They were implanted in sheep models. Unfortunately, the success of this study was limited because the polyesters were insufficiently flexible and could not function as leaflets in a tri-leaflet valve. In the late 1990s, another study was performed, this time using poly-3-hydroxyoctanoate-co-3hydroxyhexanoate (P3HO3HH). It was used as the scaffold material for the valve leaflet and subsequently the entire heart valve. Th e results were very promising. When the tissue engineered heart valves were implanted in the pulm onary circulation, the sheep survived the complete duration of the study, and the scaffolds began to remodel in vivo to resemble the native valve.61 The next improvement was to use P4HB as a faster degrading, alternative scaffold material. After implantation, in place of the native pulmonary valve, the tissue engineered heart valve functioned well, a nd echocardiography of th e implanted valves demonstrated functioning mobile leaflets wit hout any stenosis, thrombus, or aneurysm. In addition, (i) the tensile mechanical properties of the valve were almost indistinguishable from the native valve, (ii) biochemical analys is revealed a similar make up to the native counterpart, (iii) histological analysis of the leaflet stru cture showed that the new structure was made up of three distinct orga nized layers, a fibrous layer of collagen, a loose layer rich in glycosaminoglycans (GAGs) a nd a layer of elastin th at is characteristic

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65 of the native leaflet structure and (iv) the si ze of the valve had increased from 19mm at implant to 23mm at 20 weeks as the lamb had grown.62 6.7 (c) Sutures, medical textile products and bulking agents. P4HB can be stretched to 10 times its or iginal length. During the stretching process, the polymer chains become oriented, resultin g in exceptionally strong fibers. Another property that sets P4HB apart from curre nt synthetic polymers is a lower Youngs modulus equating improved handling, and a different breaking strengt h retention profile upon implantation. Low molecular weight oligom ers of P4HB have been prepared for potential use in soft tissue repair, augmen tation, and bulking/padding applications. These oligomers were prepared in solution by hydrol ysis of higher molecular weight P4HB, and along with P4HB microdispersions could pot entially be administered by injection.63 6.8 Conclusion Apart from the differences in mechan ical and thermal properties of P4HB and PGA these polymers degrade via different mechanisms that produce different results. PGA degrades rapidly by a process that involves pr imarily diffusion of water into the polymer followed by bulk hydrolysis. In large PGA implan ts this can lead to the accumulation of highly acidic degradation products that can suddenly be released resulting in severe foreign body reactions. It can also result in dramatic loss in implant strength soon after implantation. In contrast, P4HB appears to be degraded in part by a surface erosion process in vivo that results in: (i) a gradual reduction of mechanical strength in vivo and

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66 (ii) a good biocompatibility due to a slow rele ase of well tolerated le ss acidic degradation products.64 P4HB has proved itself a definite heavyw eight in the medical field. Its suitability has been assessed on its stand alone performance, and comparison to PGA and other traditional synthetics. P4HBs biocompatibility to in vivo applications, is but one weapon in an arsenal of copolymers that further extend the properties of biopolymers in this field. CHAPTER 7. Analysis of Poly-3-hydroxybutyrate (P3HB) content in Panicum virgatum (switchgrass)

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67 The second part of the thesis involve s the analysis of PHA content in switchgrass. Work done at Metabolix, a bi oengineering company based in Cambridge, Massachusetts, during summer 2005 and ISPs 2006 and 2007, wa s instrumental in developing new pathways in transgenic plants that could improve PHA production, in terms of labor, cost and time. Traditional methods of PHA production like fermentation, was efficient in some areas, like percentage PHA accumulation in bacteria cells, and polymer purity, but not in others, like cost. After having expe rimented with plants like tobacco, and Arabidopsis thalium there was a shift towards gene expression in switchgrass. The preference of switchgrass was based not only on its individual merit as a highly tolerable and resilient crop, especially with respect to nutrient demands and water provisions, but also the unfavor ability of the other competi ng host plants. In particular, the idea of genetically modifying tobacco woul d have been a contentious issue because of the large smoking population. My time at Metabolix coincided with switc hgrass research efforts. Working under the guidance of Senior Q.C Analyst, Sean Daught ry, thousands of switchgrass plants were analyzed for poly-3-hydroxybutyrate content. The highest producing batches, namely batch 10, batch 11, batch 12, batch 13, batch 14 were kept for further and future work to be done with these samples. Results at this ti me are not where they should ideally be, but in defense of switchgrass efforts, there has not been the advantage of substantial time. What has been achieved is a gradual increase in P3HB content from the first plants that were modified. Even though the percentages ar e very small, on the sc ale of thousands of hectares of switchgrass, no doubt there s hould be sufficient PHA for reducing our dependence on petroleum derived plastics and striving towards a greener future.

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68 7.1 Butanolysis Butanolysis is a primary screening tec hnique used to ascertain the PHA content of different constitutive and transgenic plant samples. The genes responsible for PHA production in microorganisms have been inco rporated into switchgrass plants using recombinant DNA technology in order to create plants with specific characteristics. From a wide selection of hundreds of switchgrass plants, routine butanolysis was conducted and those plants yielding the highest percenta ges of P3HB were save d, while the others were discarded. Of the saved plants, their profiles were considered and optimized to improve P3HB yield. 7.2 Introduction Poly-3-hydroxybutyrate (P3HB) is a polyest er that is currently being developed for many applications, in various fields, wh ere it serves a functional equivalence to traditional plastics. P3HB has natural or igins and, as such, is susceptible to biodegradation after it has se rved its purpose. In additi on, biopolymers reduce petroleum dependence, and in contrast to traditional pl astics, do away with post-usage and disposal concerns. Commercialization of P3HB ha s traditionally been done by bacterial fermentation, but with metabolic engineeri ng advancements it has become possible to harvest this polymer from plants. The huge scale and favorable economics of agriculture make plants very compelling metabolic engineer ing targets. Plants must derive all their own organic compounds from carbon dioxide in the air and the favorable economics of plants make them the ideal, low cost biofactories for natural plastics.65 In some

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69 applications where a high pe rcentage of purity and quality control is necessary, fermentation will always trump other processes. But for the majority of applications, PHA production from switchgrass is something to be pursued with vigorous interest. 7.3 Experimental Procedures and Methods. Sample preparation From a wide selection of transgenic P3HB producing plants, leaf samples averaging approximately 300mg were cut and placed into glass vials. These leaves were dried for two to four days in the lyophili zer, and were then crushed or cu t, to ensure that fragments of the sample covered the base of the vial in prepar ation for weighing. Butanolysis and GC/MS scanning. Up to 300mg of the dried sample was tran sferred to another glass vial and weighed. Several standards of pure P3HB polymer were also prepared. Three milliliters of GC/MS reagent (90% n-butanol, 10% concentrated hydrochloric acid, and diphenylmethane internal standard (INS) at 0.5mg/mL) were di spensed in each vial and the reaction was run at 110C for 3 hours. After the samples and standards were cooled, the inorganic impurities were removed with a single water extraction. The samples were then centrifuged for five minutes. A 1mL aliquot of the organic layer of each sample was added to a GC vial. The samples were analyzed on the GC/MS w ith a DB225MS column with a ramp from 80C to 230C at 8C/minute. Selected ions of the resulting ester, butyl 3-hydroxybutyrate were 87.0, 43.1 and 89.0 atomic mass units (amu). Selected ions for the internal standa rd were 168.1, 167.1 and 152.1 amu. A standard curve was prepared from the weighed standards, and the unknown ma ss of P3HB, present in each sample was

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70 calculated from the standard curve. This cal culated mass, when divided by the mass of the sample, yielded the % P3HB for each sample. Calculation consideratio ns for GCMS analysis. GCMS uses an internal standard which co rrects for discrepancies in injection volume. Analytical software is used to perform con centration calculations from chromatograms, integrated based upon peak area. The calibration curve is gene rated by a linear regression of the analytical standards. The linearity of the calibrati on curve can be confirmed, by the coefficient of variation of the lin ear regression being greater than 0.99. In comparison to software, analytical methods, a manual calculation may be done by creating a calibration curve of amount ratio, pl otted on the abscissa, versus response ratio, plotted on the ordinate. The am ount ratio is the P3HB c oncentration divided by the diphenylmethane internal standard concentratio n. The response ratio is the P3HB area divided by the diphenylmethane area. The re sponse factor, F, is found by solving the following equation, which is the grad ient of the standard curve. The workings of the GCMS is very elaborate but as far as its relevance to this thesis, it is important to understand what exactly is occurring. Each unknown sa mple is injected into the GC, in combination with an internal standard. To correct for discrepancies in injection volumes of each sample, the area of the diphenylmethane peak is corrected, or normalized to the injection of the blank sample In any run, if the area of the internal standard peak is less than that of the blank, then a smaller volume of sample was injected and the observed area must be increased. On the other hand, if the area of the internal signal INS of Area signal P3HB of Area = F { ]INS[ ]P3HB[ } Equation (1)

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71 standard peak is greater than the blank, then the area must be decrea sed to correct this excess. The volume correction factor is the ratio of the internal standard area in the sample to the area of the internal standard peak in the reference sample. This volume correction factor is then used to corre ct the area of the unknown component to a consistent scale. The areas of the P3HB signal as well as the INS signal are obtained from the GC, and their ratio is calculated as the response ra tio. From equation 1, the response factor is multiplied by the amount ratio which is obtained from the MS. The GC separates the analyte and identifies each eluted component to obtain the concentration of target ions with specific mass to charge (m/z) ratios. Each scan involves a tabulated list of the mass detector's signal for each m/z fragment All of the intensities of each scan are summated as the total ion count (TIC) versus time. It would be noticed from the standard curv es, that each standard mass, corresponds to an amount ratio of twice the standard mass. For example, looking at batch 10, vial 56 had a standard mass of 0.2mg P3HB. But, since amount ratio is defined as and [INS] was 0.5mg/ml, vial 56 had a value of 0.2/0.5 = 0.4 amount ratio units. Because the concentration of the inte rnal standard was the same throughout all the samples, each weighed P3HB standard had a coordinate on the horizontal axis which was numerically equivalent to twice its mass. This shifting of the standard curve along the x-axis by a scale factor of 2, did not affect the dete rmination of the amount ratio by GCMS. After drawing the standard curve, unknown samples were subjected to GCMS analysis, and a TIC was produced. Using equa tion 1 or software programming, this TIC was used to determine the corresponding amount of P3HB for a distinct response ratio.

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72 This was done by reading off the x value, at the intersection of the y value and the generated standard curve. Figure 7.1 is illustrative fo r data acquisition purposes. Figure 7.1Standardized curve em ploying butanolysis to find [P3HB in an ideal situation where slope=1. 7.5 Results A total of 273 transgenic P3HB produc ing switchgrass plants were scanned using the GCMS, as detailed in the experimental sect ion. Graphs and Excel sheets follow, with each excel sheet being supplemented by a standa rd curve for that batch. Batch 11 had the highest percentages, with samples 155-11 and 155-17 yielding 0.394% and 0.083% P3HB respectively, while batch 10 had the lowest sample highs, of 0.005% and 0.004%. Table 7.1 shows a breakdown of the polymeri c content in samples, by batch, in increments of 0.002 percentage units. The two highest producing samples, per batch is recorded in table 7.2. From thes e results, one thing is clear. GCMS is reliable analytical method for screening switchgrass samples for PHA content. Intervals Batch 10 Batch 11 Batch 12 Batch 13 Batch 14 Frequency Frequency Frequency Frequency Frequency

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73 0.0-0.2 48 34 30 33 48 0.2-0.4 5 12 15 2 0.4-0.6 1 4 3 0.6-0.8 2 4 2 3 3 0.8-1.0 2 2 1 1.0-1.2 2 1.2-1.4 5 1 2 1.4-1.6 1 1.6-1.8 1 1.8-2.0 3 2.0-2.2 1 2.2-2.4 2.4-2.6 1 2.6-2.8 2.8-3.0 3.0-3.2 3.2-3.4 1 3.4-3.6 3.6-3.8 3.8-4.0 >4.0 2 Table 7.1 Range of polymeri c content of batches 10 thro ugh 14, by 0.002% increments. From table 7.1, the greatest proportion of polym er is contained in the (0.0-0.2) range for the five batches. Results in this range are quite common for the unreported batches, where low polymeric yields were recorde d. For the five batches under investigation, however, it is clear that higher polymeric contents are pres ent and are spread nicely, especially in batches 11, 12, over a good range. In some batches, like batch-11, 0.39% is a definite outlier. Nevertheless, this does not deride the fact that this batch had 22% of its samples producing yields in the 0.012 range. A similar, but less dense distribution was seen in the high end range for batch12. Batches 10, 13 and 14 were observed to contain greater frequency densities in the low end range, with batches 11 and 14 having the exact freque ncy in the 0.0-0.2 range, but differing slightly from the 0.6-0.8, to the 1.2 -1.4 interval. See Figure 7.3.

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74 These results will be further analyzed and their implications considered in the discussion section. BATCH SAMPLE %P3HB 10 2I-160 0.005 10 2I-119 0.004 11 155-11 0.394 11 155-18 0.083 12 2I-195 0.020 12 2I-210 0.017 13 2I-126 0.007 13 2I-266 0.009 14 155-2 0.014 14 155-3 0.013 Table 7.2Highest producing P3HB samples for batches 10 through 14. Figure 7.2 Diagrammatical comparison/ representation of batch frequencies.

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75 Figure 7.3 -Target ions for diphenylmethane in ternal standard. The first diagram shows the abundance as a function of time, while the second shows the abundance as a function of m/z ratio.

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76 Figure 7.4 -Target ions for butyl-3-hydroxybutyrate. The first diagram shows the abundance as a function of time, while the second shows the abundance as a function of m/z ratio.

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77

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78 Figure 7.5-Standard curve for batch 10 of Response Ratio versus Amount Ratio.

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79

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80 Batch 10 vial sample Sample Mass/ mg P3HB mass/mg %P3HB 1 2I-149 38.5 0 0.000 2 2I-150 24.1 0.001 0.004 3 2I-151 67 0 0.000 4 2I-152 33.6 0.001 0.003 5 2I-153 34.1 0.001 0.003 6 2I-154 35.7 0.001 0.003 7 2I-155 21 0 0.000 8 2I-156 59.9 0 0.000 9 2I-157 9.8 0 0.000 10 2I-158 27.1 0 0.000 11 2I-159 54.8 0 0.000 12 2I-160 19.6 0.001 0.005 13 2I-161 26.3 0 0.000 14 2I-162 30.3 0 0.000 15 2I-163 60 0 0.000 16 2I-164 92 0 0.000 17 2I-165 42.6 0 0.000 18 2I-166 41.5 0 0.000 19 2I-167 55.9 0 0.000 20 2I-168 46.4 0 0.000 21 2I-169 8.9 0 0.000 22 2I-170 68.6 0 0.000 23 2I-171 42.3 0 0.000 24 2I-172 30.5 0.001 0.003 25 2I-173 95.9 0 0.000 26 2I-174 64.2 0 0.000 27 2I-175 85.8 0.001 0.001 28 2I-176 59.2 0.001 0.002 29 2I-177 49.2 0 0.000 30 2I-178 48 0.001 0.002 31 2I-179 53.7 0 0.000 32 2I-180 48.1 0 0.000 33 2I-181 89.3 0 0.000 34 2I-182 34.1 0 0.000 35 2I-183 46.7 0 0.000 36 2I-184 33.2 0 0.000 37 2I-109 26 0 0.000 38 2I-110 25.8 0 0.000 39 2I-111 13.2 0 0.000 40 2I-112 30.9 0 0.000 41 2I-113 28.2 0 0.000 42 2I-114 28.2 0 0.000 43 2I-115 37.6 0 0.000 44 2I-116 11.5 0 0.000 45 2I-117 18.5 0 0.000 46 2I-118 10.5 0 0.000 47 2I-119 16.1 0.001 0.006 48 2I-120 14.9 0 0.000 49 2I-121 15.5 0 0.000 50 2I-122 43.9 0 0.000 51 2I-123 29.4 0 0.000 52 2I-124 26.1 0 0.000 53 PCAM52 24.1 0 0.000 54 PCAM53 13.9 0 0.000 55 PCAM54 9.4 0 0.000 Vial Standards Mass/mg P3HB mass/mg %P3HB 56 P3HB1 0.2 0.53 265.000 57 P3HB2 0.4 0.68 170.000 59 P3HB3 2.5 2.43 97.200 60 BLANK 0 0 0 Table 7.3Data for batch 10.

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81 Figure 7.6Standard curve for batch 11 of Response Ratio versus Amount Ratio.

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82 Batch 11 vial sample Sample Mass/mg P3HB mass/mg %P3HB 1 155-10 8 0.001 0.013 2 155-11 3.3 0.013 0.394 3 155-12 10.6 0.002 0.019 4 155-13 20.9 0.004 0.019 5 155-14 10.6 0 0.000 6 155-15 5.8 0 0.000 7 155-16 12.9 0 0.000 8 155-17 3.8 0.001 0.026 9 155-18 1.2 0.001 0.083 10 155-19 6 0.002 0.033 11 155-20 15.8 0.001 0.006 12 155-21 6.7 0.001 0.015 13 155-22 7.8 0 0.000 14 155-23 11.1 0 0.000 15 155-24 7.8 0.001 0.013 16 155-25 8.2 0.001 0.012 17 155-26 10.3 0 0.000 18 155-27 3.6 0 0.000 19 155-28 5.1 0 0.000 20 155-29 7.6 0 0.000 21 155-30 5.2 0.001 0.019 22 155-31 10.9 0 0.000 23 155-32 7.4 0.001 0.014 24 155-33 11.3 0.001 0.009 25 155-34 6.1 0 0.000 26 155-35 5.8 0 0.000 27 155-36 9.9 0.001 0.010 28 155-37 16.6 0 0.000 29 155-38 15.1 0.001 0.007 30 155-39 7.3 0.002 0.027 31 155-40 17.8 0 0.000 32 155-41 7.2 0.001 0.014 33 155-42 7.8 0.001 0.013 34 155-43 8.9 0 0.000 35 155-44 5.3 0 0.000 36 155-45 10.3 0 0.000 37 155-46 15.6 0.001 0.006 38 155-47 14.6 0 0.000 39 155-48 8.5 0 0.000 40 155-55 6.1 0 0.000 41 155-56 12.6 0 0.000 42 155-58 3.9 0 0.000 43 155-59 7.5 0 0.000 44 155-60 19.6 0.001 0.005 45 155-61 8.3 0 0.000 46 155-68 13.1 0 0.000 47 155-69 8.8 0 0.000 48 155-70 8.4 0 0.000 49 155-71 3.2 0 0.000 50 155-72 14 0 0.000 51 155-73 8.8 0 0.000 52 155-87 10.5 0 0.000 53 155-88 7.3 0 0.000 54 155-89 11 0 0.000 55 PCAM 6 12.6 0 0.000 Vial Standards Mass/mg P3HB mass/mg %P3HB 56 P3HB1 0.5 0.71 142.000 57 P3HB2 2 1.84 92.000 58 P3HB3 2.3 2.1 91.304 59 P3HB4 3.79 3.79 100.000 60 BLANK 0 0 0 Table 7.4Data for batch 11.

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83 Figure 7.7-Standard curve for batch 12 of Response Ratio versus Amount Ratio.

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84 Batch 12 vial sample Sample Mass/mg P3HB mass/mg %P3HB 1 155-10 8 0.001 0.013 2 155-11 3.3 0.013 0.394 3 155-12 10.6 0.002 0.019 4 155-13 20.9 0.004 0.019 5 155-14 10.6 0 0.000 6 155-15 5.8 0 0.000 7 155-16 12.9 0 0.000 8 155-17 3.8 0.001 0.026 9 155-18 1.2 0.001 0.083 10 155-19 6 0.002 0.033 11 155-20 15.8 0.001 0.006 12 155-21 6.7 0.001 0.015 13 155-22 7.8 0 0.000 14 155-23 11.1 0 0.000 15 155-24 7.8 0.001 0.013 16 155-25 8.2 0.001 0.012 17 155-26 10.3 0 0.000 18 155-27 3.6 0 0.000 19 155-28 5.1 0 0.000 20 155-29 7.6 0 0.000 21 155-30 5.2 0.001 0.019 22 155-31 10.9 0 0.000 23 155-32 7.4 0.001 0.014 24 155-33 11.3 0.001 0.009 25 155-34 6.1 0 0.000 26 155-35 5.8 0 0.000 27 155-36 9.9 0.001 0.010 28 155-37 16.6 0 0.000 29 155-38 15.1 0.001 0.007 30 155-39 7.3 0.002 0.027 31 155-40 17.8 0 0.000 32 155-41 7.2 0.001 0.014 33 155-42 7.8 0.001 0.013 34 155-43 8.9 0 0.000 35 155-44 5.3 0 0.000 36 155-45 10.3 0 0.000 37 155-46 15.6 0.001 0.006 38 155-47 14.6 0 0.000 39 155-48 8.5 0 0.000 40 155-55 6.1 0 0.000 41 155-56 12.6 0 0.000 42 155-58 3.9 0 0.000 43 155-59 7.5 0 0.000 44 155-60 19.6 0.001 0.005 45 155-61 8.3 0 0.000 46 155-68 13.1 0 0.000 47 155-69 8.8 0 0.000 48 155-70 8.4 0 0.000 49 155-71 3.2 0 0.000 50 155-72 14 0 0.000 51 155-73 8.8 0 0.000 52 155-87 10.5 0 0.000 53 155-88 7.3 0 0.000 54 155-89 11 0 0.000 55 PCAM 6 12.6 0 0.000 Vial Standards Mass/mg P3HB mass/mg %P3HB 56 P3HB1 0.5 0.71 142.000 57 P3HB2 2 1.84 92.000 58 P3HB3 2.3 2.1 91.304 59 P3HB4 3.79 3.79 100.000 60 BLANK 0 0 0 Table 7.5Data for batch 12.

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85 Figure 7.8-Standard curve for batch 13 of Response Ratio versus Amount Ratio.

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86 Batch 13 vial sample Sample Mass/mg P3HB mass/mg %P3HB 1 2I-125 23.1 0.001 0.004 2 2I-126 30.3 0.002 0.007 3 2I-127 40.2 0 0.000 4 2I-128 31.3 0 0.000 5 2I-129 40.2 0.001 0.002 6 2I-306 28.1 0.001 0.004 7 2I-307 38 0.001 0.003 8 2I-308 59.5 0.002 0.003 9 2I-309 70.9 0.002 0.003 10 2I-310 57 0.002 0.004 11 2I-311 53.5 0.004 0.007 12 2I-312 46.2 0.001 0.002 13 2I-313 37.3 0.002 0.005 14 2I-240 92.1 0.003 0.003 15 2I-241 65.3 0.001 0.002 16 2I-242 47.9 0.001 0.002 17 2I-243 52.8 0.001 0.002 18 2I-244 41 0 0.000 19 2I-245 35.6 0.001 0.003 20 2I-246 40.6 0 0.000 21 2I-247 53.5 0 0.000 22 2I-248 43.3 0 0.000 23 2I-249 22.6 0 0.000 24 2I-250 82.1 0 0.000 25 2I-251 90.9 0.001 0.001 26 2I-252 54.6 0 0.000 27 2I-253 56.1 0 0.000 28 2I-254 33.3 0.002 0.006 29 2I-255 30.1 0.001 0.003 30 2I-256 36.4 0 0.000 31 2I-257 34.75 0.001 0.003 32 2I-258 56.8 0.001 0.002 33 2I-259 23.9 0 0.000 34 2I-260 21.4 0.001 0.005 35 2I-261 52.7 0 0.000 36 2I-262 32 0.001 0.003 37 2I-263 24.6 0 0.000 38 2I-264 28.6 0 0.000 39 2I-265 45.1 0.002 0.004 40 2I-266 22.1 0.002 0.009 41 2I-267 25.5 0.001 0.004 42 2I-268 15.4 0 0.000 43 2I-269 30.4 0.001 0.003 44 2I-270 41.9 0.001 0.002 45 2I-271 34.5 0.001 0.003 46 2I-272 17.9 0 0.000 47 2I-273 26.3 0 0.000 48 2I-274 10.4 0 0.000 49 2I-275 44.2 0.001 0.002 50 PCAM44 105.4 0.001 0.001 51 PCAM45 86.4 0 0.000 52 PCAM46 122.1 0.001 0.001 53 PCAM47 37.9 0.002 0.005 54 PCAM48 148.9 0.003 0.002 55 PCAM49 96.5 0.002 0.002 Vial Standards Mass/mg P3HB Mass/mg %P3HB 56 P3HB1 0.2 0.29 145.000 57 P3HB2 0.9 0.87 96.667 58 P3HB3 2 2.31 115.500 59 P3HB4 3 2.79 93.000 60 BLANK 0 0 0 Table 7.6Data for batch 13.

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87 Figure 7.9-Standard curve for batch 14 of Response Ratio versus Amount Ratio.

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88 Batch 14. vial sample Sample Mass/mg P3HB mass/mg %P3HB 1 155-1 10.4 0.001 0.010 2 155-2 21.9 0.003 0.014 3 155-3 22.9 0.003 0.013 4 155-4 23.5 0.001 0.004 5 155-5 13.1 0 0.000 6 155-6 8.2 0 0.000 7 155-7 9.4 0 0.000 8 155-8 2.4 0 0.000 9 155-9 4.1 0 0.000 10 155-49 26.8 0 0.000 11 155-50 8.7 0 0.000 12 155-51 7.3 0 0.000 13 155-52 7.6 0 0.000 14 155-53 13.7 0 0.000 15 155-54 13.6 0.001 0.007 16 155-62 10 0 0.000 17 155-63 14.3 0.001 0.007 18 155-64 12.5 0 0.000 19 155-65 15.1 0.001 0.007 20 155-66 1.7 0 0.000 21 155-67 11.9 0 0.000 22 155-74 11.4 0 0.000 23 155-75 9 0 0.000 24 155-77 17.6 0 0.000 25 155-78 4.5 0 0.000 26 155-80 13.5 0 0.000 27 155-81 11.8 0 0.000 28 155-82 8.3 0 0.000 29 155-83 3 0 0.000 30 155-84 11.5 0 0.000 31 155-85 10.6 0 0.000 32 155-86 13 0 0.000 33 155-90 3.8 0 0.000 34 155-91 11.2 0 0.000 35 155-92 10 0 0.000 36 155-93 11.9 0 0.000 37 155-94 12.3 0 0.000 38 155-95 13.2 0 0.000 39 155-96 12.6 0 0.000 40 155-97 9.3 0 0.000 41 155-98 5.4 0 0.000 42 155-99 15.2 0 0.000 43 155-100 6.7 0 0.000 44 3I-314 11.8 0 0.000 45 3I-315 41.3 0 0.000 46 3I-316 37.8 0 0.000 47 3I-317 58.3 0.001 0.002 48 3I-318 97.5 0.001 0.001 49 3I-319 108.8 0.002 0.002 50 3I-320 88.1 0.001 0.001 51 3I-321 47.8 0 0.000 52 3I-322 43.2 0 0.000 53 3I-323 33.7 0.001 0.003 54 3I-324 90.7 0.002 0.002 55 3I-325 54.9 0 0.000 Vial Standards Mass/mg P3HB Mass/mg %P3HB 56 P3HB1 0.1 0.23 230.000 57 P3HB2 1.4 1.28 91.429 58 P3HB3 2.3 2.19 95.217 59 P3HB4 4 4.1 102.500 60 BLANK 0 0 0 Table 7.7Data for batch 14.

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89 7.5 Discussion This section will highlight the mechanism of butanolysis, as well as sources of error and precautionary measures, employed to achieve re asonable accuracy and precision during all parts of the experimental design. The pathway of P3HB synthesis in A. Eutropus will be explained followed by a section on the economic consid erations of PHA production in plants. 7.5(a) Chemistry of Butanolysis Butyl-3-hydroxybutyrate, as well as butyl -3-chlorobutyrate are the main products of butanolysis (Figure 7.10). Other ions from th ese products, present in the mass spectrometer report, were removed using GCMS signal optimization. Only the major ion peaks are considered. Figure 7.10 (a) In the first step, P3HB abstracts a hydrogen ion from solu tion. The proton becomes attached to one of the lone pairs on the double bonded oxyge n. Protonation of the ester carb onyl makes it more electrophilic Figure 7.10 (b) The oxonium ion forms a stable canonical form (a carbocation) in prepar ation for attack by butanol. Figure 7.10 (c) The butanol O functi ons as the nucleophile attacking the carbocation, creating a tetrahedral intermediate.

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90 Figure 7.10 (d) Proton transfer occurs from a butanolic O to an adjacent, ( OR) oxygen. This is facilitated by a substance in the mixture that basically acts as a transfer agent, via a proton pi ck up, and dump mechanism. The adjacent (-OR) group is now protonated, and is a good leaving group. Figure 7.10 (e) A molecule (HO-R) is lost from the ion. A carbocation is regenerated. Figure 7.10 (f) The hydrogen is removed from the oxygen by reaction with a water molecule. The product butyl-3hydroxybutyrate is formed, when R = H. At this point, nucleophilic substitution of Cl for OR may occur, forming butyl-3chlorobutyrate. This however, is a minor product. Figure 7.10 Mechanism of Butanolysis. 7.5(b) Sources of experimental error and precautionary measures Butanolysis, although an accurate analytical tech nique is not without random and systematic error. The challenges of weighi ng P3HB standards for the sta ndard curve were indexed by inconsistencies in their weighed values when ch ecked alongside their experimentally calculated values. Attempts were made to reduce er ror by using different balances and having measurements made by different analysts. The scale used for weighing was an Ohaus Explorer PRO analytical balance with a 0.1mg readability for maximum accuracy. In addition gloves were used minimize fingerprints and moisture, while draftshields were us ed to nullify eddy and wind currents. Finally anti-static guns were used to prevent dust to sample attraction. The accuracy of the individual weighings we re judged against the GCMS determination of

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91 the actual quantity of P3HB present in each of th e four standards. In batch 10 for example, vials 56 and 57 had errors of 165% and 70% respectively, between th e weighed and experimental values. Batch 11 was slightly more accurate with a maximum error in vial 56 of 29%, and an average error in the 3 most accura te samples of 4%. Batch 12 was almost perfect except with an average error of 4% for the most accurate sample s, except for an underestimate in vial 56 of 50%. Batch 13 was similar to batch 11, wi th an average error of 2% for the most accurate samples, and a maximum error of 45%. Finally, batch 14 had an error of 4% for the most accurate samples, with a large error of 130% in vial 56. Analysis of the weighed standards confirms the conclusion that scale sensitivity, and accuracy increases for higher masses. Though it may be feasible to use higher masses to define the standard curve, the masses of our P3HB samples are in the lower end of the range. A suggested alternative for determin ing the standard curve would have been to use serial dilution instead of individual weighings. It is common in immunoassays to have a single stock and do serial 1:2 dilutions. Initially one ends up with which is then diluted to 1:2. This dilution gives from the primary stock. This is diluted 1:2 ag ain and gives 1/8 from the primary stock etc. From the primary stock one has dilutions of , etc. which is good for an immunoassay requiring at least a two log concentration range. Within the context of the required specifi cations for butanolysis, there are three main drawbacks to serial dilution. The first is that any dilution error made in the first dilution will propagate through the whole series. Such an error would be compounded for each subsequent dilution from the stock. Secondly, in serial dilution, all standards are differe nt by a scalar consta nt (k). In the case described above, where k=2, for curve fitting requi rements, this will overweigh the less dilute

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92 dilutions and underweigh the more dilute dilutions In general the more d ilute dilutions are less precise, so they should actually have equal or great er weight as this will increase th e error in more dilute samples, which are often the most critical, especially in contaminant assays. An arithmetic dilution series will more evenly spre ad out the dilution concen trations and more than one will be made from the primary stock. By having more evenly spaced standards, there is better comparability between the different dilutions wh ich better approximates the function used for calculations. The third factor working against serial d ilution is the limited qua ntities of pure P3HB polymer. The worlds supply at this time (appr oximately 25g) is insufficient for the large quantities of pure polymer required for accurate molalities (large mass of polymer per unit mass of solvent) and subsequent dilution. In analytical experiments like butanolysis, repetition is a dvisable and should be encouraged, to reduce random error. The only drawback is the fact that GCMS analysis of 156 samples takes as much as two days to be performed. Scanning in triplicate could be a hindrance with the sheer bulk of new plants constantly being produced for analysis while striving to maintain internal deadlines for report analysis. Furthermore, re peating a preliminary sc reening technique is impractical, given that plant samples expressing high levels polymer are immediatelytaken for enhancement by the botany group. 7.5(c) History of PHA: Bacterial PHA Biosynthetic Pathways. In Alcaligens eutropus P3HB is synthesized from acetyl-CoA by the sequential action of three enzymes (Figure 7.2). The first enzyme is 3ketothiolase and catalyzes the formation of acetoacetyl-CoA from acetyl-CoA. Acetoacetyl-CoA reductase then reduces Acetoacetyl-CoA to

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93 R-(-)-3-hydroxybutyryl-CoA, which is then polyme rized by PHA synthase to form P3HB. When A. eutropus is fermented in the presence of a high glucose content, but limited in one essential nutrient, P3HB granules can accumulate and act as carbon reserves in the bacteria. P3HB inclusions can be as much as 80% of cell dry weight. 66 Figure 7.11Pathway of PHB synthesis in A. Eutropus. PhbA, PhbB and PhbC sequentially act on acetyl-CoA and its product to form PHB. In comparison to bacterial or yeast fermen tation, crop plants are cap able of producing large amounts of a number of useful chemicals at low cost. With recent advances in plant molecular biology allowing expression of fore ign proteins in a variety of plants, genetic engineering of crops has allowed scientists to increase the quan tity and modify the type of products produced in crop plants.67 Since pharmaceutical products are typically of high value, are very pure and are required in relatively small amount s (ie. kg or a few tons), it is unlikely that their production in plants could compete with fermentation or expr ession in transgenic an imals. However products needed in large quantities and at relatively lo w purity, may potentially be produced commercially in plants. 7.5(d) Economics of PHA development in plants In view of the flexibility of plants in expres sing foreign proteins, it was of interest to explore the possibility of synthesizing PHAs in plants Production of PHA on an agricultural scale could

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94 allow synthesis of biodegradable plastics in the million ton scale compared to fermentation which produces material in the thousand ton scale. Furthe rmore, if PHA can be synthesized in plants to a level comparable to 20 % dry weight, PHA coul d potentially be produced at a cost of $0.60/kg and, thus, be competitive with petroleum-derived plastics. 7.6 Microbial Gene Expression in Plants This section discusses the transgenic procedures for the incorporation of bacterial PHA producing genes in germinating plants. It gives a brief history of the work done on Arabidopsis thaliana, and the engineering ingenuity of expressing no n-endogenous proteins in the plastid cells of A. thaliana. This redirected pathway resulted in a 100 fold increase in P3HB production in comparison to the cytoplasmic cells of the plant sample. 7.6(a) Cytoplasmic Expression Synthesis of PHB in plants was initially explored by expression of the PHB biosynthetic genes of the bacterium Alcaligens eutropus in the plant Arabidopsis thaliana .68 Although of no agricultural importance, A. thaliana was chosen because of its extensive use as a model system for genetic and molecular studies of plants. Of the three enzymes required for PHB synthesis from acetyl-CoA, only the 3-ketothio lase is endogenously present in plants. In order to complete the PHB pathway in plants, the A. eutropus genes encoding the acetoacetyl-CoA reductase and PHA synthase were expressed in transgenic A. thaliana under the control of the constitutive CaMV 35S promoter69. Since the bacterial proteins were not modi fied for targeting to any particular subcellular component, the foreign proteins we re expected to be expressed in the cytoplasm. Transgenic A.

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95 thaliana hybrids expressing the reductase and PHA s ynthase produced low amounts of PHB as detected by GCMS. The maximum amount detected was approximately 0.14 % dry weight. This yield was two orders of magnitude less than the target amount for viable commercial production in plants.70 7.6(b) Plastid Expression Worth mentioning is the fact that when the foreign proteins were expressed in the plastids of A. thaliana significant increases in % P3HB were obtaine d. Biosynthesis of fa tty acids in plants accumulates in the plastid, starting from acetyl-CoA. This accumulation is more enhanced in the seed of oil-storage plants like A. thaliana, where up to 40% of seed dr y weight is triglycerides.71 See Figures 7.13 and 7.14. It was surmised that the small proporti ons of Acetyl-CoA in th e cytoplasmic cells of A. thaliana was responsible for the low P3HB yields (0.14% dry weight). Si nce the Acetyl-coA concentration is much highe r in the plastid cells of A. thaliana it was hypothesized that enzyme expression in these parts could le ad to higher yields of P3HB.72 Expression of the P3HB biosynthetic pathway in the plastid cells was achieved by modification of phaA, phaB and phaC for plastid targeting. Each enzyme wa s expressed under CaMV 35S promoter. Analysis revealed that P3HB inclusions accumulated exclusively in the plastids. The P3HB content gradually increased over the lifespan of the plant, with fully expanded pre-senescing leaves typically showing ten times more P3HB than younger expa nding leaves of the same plant. P3HB percentages were determined at a maximum of 14% dry weight.73 A. thaliana s 100 fold increase in P3HB production achieved by redire cting the biosynthetic pathway seems very inspiring. Plastid

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96 threonine propionyl-CoA acetyl-coA fatty acids ilvA PDC btkBRe phaARe 2-ketobutyrate 3-ketovaleryl-Co A acetoacetyl-CoA phaBRe phBRe R-3-hydroxyvaleryl-CoA R-3-hydroxybutyryl-CoA phaCRe phaCRe isoleucine P(HB-HV) PHB Acetyl-coA Acetoacetyl-CoA malonyl-CoA 3-hydroxybutyryl-coA phaCRe Isoprenoids PHB flavonoids Figure 7.13 Modification of plant metabolic pathways for the synthesis of P3HB and P3HB3HV. Pathways created by the expression of transgen es are in green, while endogenous pl ant pathways are in plain letters. The various transgenes expressed in plants are in italics. 7.14 Accumulation of P3HB granule inclusions in chloroplasts of A. thaliana, after transgenic expression in the plastid cells. Note arrows. 7.7 Putting Switchgrass Analysis in context. PHA production is switchgrass is an index of the a dvances in metabolic engineering practices in green plants. Switchgrass is an attractive host for PHA production because it is a leading candidate for bio-based energy production it grows in high yield, can be grown on land of marginal use for other crops, and fixes about 2kg carbon dioxide in its root system for every kg C y to p lasm

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97 of biomass above the ground. Yields average a bout 5 tons per acre in the U.S today, and experimental plots have demonstr ated yields of about 10 tons pe r acre over a six year period. It can be expected that application of the tools of modern biotechnology w ill enhance these yields further.74 In addition the celluosic waste generated coul d be used to generate energy for driving process reactors, and this could improve the economics of the entire process. This translates well for the consumer. The results from A. thaliana in 1995 were 0.14% dry weight P3HB accumulation. However, analysis done on switchgrass in summer 2005, de tected amounts of 0.394% dry weight. This is undoubtedly three times as hi gh, and more encouraging is the fact that switchgrass has only been exposed to two years of intensive study. Even though the target is still very far away, it seems that achieving at least whole number percents may be possible if gene expression is carried out in other areas of the plant where accumulation may be more favorable. Though switchgrass is not an oil crop, there is the potential for increased yields via a similar redirected biosynthetic pathway. Even a 10 fold increase compared to A. thalianas 100 fold increase as a result of plastic expression instead of cytoplasmic, could boost P3HB percentages to whole numbers (0.39% to 3.9%). With whole numbers, more accurate results could be determined in terms of the standardized curve and the associated sources of error could be reduced tremendously. This is because standa rd weighings would be done for higher P3HB masses, since the expected yields in unknown samples would be greater. 7.8 Conclusion Within the limits of experimental error, but anolysis has proved to be an effective screening technique for polymer presence in transgenic sw itchgrass samples. GCMS analysis has revealed

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98 batch 11 as the most promising. Efforts aime d at increasing %P3HB yields will require specialized work where conditions of growth, locus and degree of ge netic expression are a few of many parameters to be considered. Further wo rk is left up to the botany and metabolic engineering team.

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99 REFERENCES 1Stevens, E.S. (2002) Green Plastics Princeton, NJ: Princeton University Press. Levy, Geoffrey. (1999) Packaging, Policy and the Environment, Aspen Publishers, Inc.3 Astle M. (1953), Organic chemistry Harper 4 Judy Brewer, History of polymers from http://matse1.mse.uiuc.edu/polymers/time.html 5 American Chemistry council, (2007 ) from www.plasticsresource.org. 6 Pryde, E. and Rothfus, J., "Industrial and Nonfood Uses of Vegetable Oils," Oil Crops of the World 7 Applied composites, corp. (2007) from http://appliedcompositescorp.birkey.com/overview.asp 8 Vacuum forming, (2008) from Wikipedia. 9 How is plastic, American Chemistry Council from http://www.plasticsresource.com/s_plasticsresource/ 10 Hornback, J. (2005) Organic chemistry Brooks Cole 11 Encyclopedia Britannica online, (20 07) Major industrial polymers from www.britannica.com/eb/article-7648 2/major-industrial-polymers 12 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Prince ton University Press. 13 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Prince ton University Press. 14 Spiro, T. and Stigliani, W. (2002) Chemistry of the Environment, Princeton, NJ: Princeton University Press.

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100 15Online article, 2007 http://www.freepatentsonline.com/3676401.html Short, William H., Environmentally disintegrable plasti c compositions comprising copolymers of ethylene and carbon monoxide a nd a degradation accelerator. 16 Online article, 2007 http://www.freepatentsonline.com/3676401.html Short, William H., Environmentally disintegrable plasti c compositions comprising copolymers of ethylene and carbon monoxide a nd a degradation accelerator. 17 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Prince ton University Press. 18 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Prince ton University Press. 19 Spiro, T. and Stigliani, W. (2002) Chemistry of the Environment, Princeton, NJ: Princeton University Press. 20 Metabolix.com (2007) http://www.metabolix.com/publications/articles.html 21 Lee Ym, et al. (2007). "Estrogen receptor independent neurotoxic mechanism of bisphenol A, an environmental estrogen. (Abstract). J Vet Sci. 8 (1): 27-38. 22 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Prince ton University Press. 23 Jordan, Shirley, (2001), Mayan Civilization: Moments in History, Perfection Learning 24 Yves Poirer, Production of polyhydroxyalkanoate s, a family of biodegradable plastics and elastomer, in bacteria and plants, Biotechnology volume 13 25 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Prince ton University Press. 26 Biotechnology Foundation: Metabolic Engineering (2006) from http://www.metabolix.com/biotechnol ogy%20foundation/metabolicengineering.html 27 Biotechnology Foundation: Metabolic Engineering (2006) from http://www.metabolix.com/biotechnol ogy%20foundation/metabolicengineering.html 28 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Prince ton University Press.

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101 29 Nynatur (2002) from http://www.nynatur.dk/english/janine_benyus_eng.html 30 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Prince ton University Press. 31Morrison, T. (1975) Organic Chemistry, Allyn and Bacon. 32 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Prince ton University Press. 33 Astle M. (1953) Organic chemistry Harper 34SWICOFIL (2007) from http://www.swicof il.com/products/212m ilk_fiber_casein.html 35 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Prince ton University Press. 36 The history of soybeans from http://www.ncsoy.org/History_of_Soybeans/history_of_soybeans.htm 37 Rimpex Rubber, China (2002) http ://www.rubberimpex.com/GuttaPercha.htm 38 The History of Plastics from www.americanchemistry.com/s_plas tics/doc.asp?CID=1102&DID=4665 39 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Princeton University Press. page 108 40 Bower D. (2005) An introduction to Polymer Physics Cambridge, Cambridge University Press from assets.cambridge.org/97805216/ 31372/excerpt/9780521631372_excerpt.pdf 41 Morrison, T. (1975) Organic Chemistry, Allyn and Bacon. 42 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Princeton University Press. page 117 43 Morrison, T. (1975) Organic Chemistry, Allyn and Bacon. 44 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Princeton University Press. page 113

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102 45 McChristy, N. Biodegradable Plas tics, The Recycler (2004), from www.metabolix.com/publications/articles.html 46 Biodegradable Plastics Developments and Environmental Impacts (2002) from www.environment.gov.au/settlements/publicatio ns/waste/degradables /biodegradable/cha pter2.html 47 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Princeton University Press page 120 48 Production of polyhydroxyalkanoates, a fam ily of biodegradable plastics and elastomer, in bacteria and plants Yves Poirer, Biotechnology, volume 13. 49 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Prince ton University Press 50 Bilby Gerald (2007) ICMA from www.icma.com/info/polymers.htm 51 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Princeton University Press page 97 52 Poirier, Y. Dennis (1992) Polyhydroxybutyrate, a biodegradable thermoplastic, produced in transgenic plants. Science 256: 520-553 53 Martin and Williams, (2000) Medical App lications of poly-4-hydroxybutyate: a strong flexible absorbable material, Biochemical Engineering Journal 54 Williams & Peoples, (1996) "Biodegradable plastics from plants," 26:38-44 Chemtech 55 Gagnon, et al., (1992). A thermoplastic elastomer produced by the bacterium Pseudomonas oleovarans, Rubber World 207:32-38 56 Martin and Williams, Applications of PHAs in Medicine and Pharmacy retrieved from http://www.tepha.com/media/phaappmp.pdf 57 Martin and Williams, (2000) Medical App lications of poly-4-hydroxybutyate: a strong flexible absorbable material, Biochemical Engineering Journal

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103 58 Martin and Williams, (2000) Medical App lications of poly-4-hydroxybutyate: a strong flexible absorbable material, Biochemical Engineering Journal 59 Martin and Williams, (2000) Medical App lications of poly-4-hydroxybutyate: a strong flexible absorbable material, Biochemical Engineering Journal 60 Martin and Williams, App lications of PHAs in Medicine and Pharmacy(2007) retrieved from http://www.tepha.com/media/phaappmp.pdf 61 Martin and Williams, App lications of PHAs in Medicine and Pharmacy(2007) retrieved from http://www.tepha.com/media/phaappmp.pdf 62 Martin and Williams, (2000) Medical App lications of poly-4-hydroxybutyate: a strong flexible absorbable material, Biochemical Engineering Journal 63 Martin and Williams, App lications of PHAs in Medicine and Pharmacy(2007) retrieved from http://www.tepha.com/media/phaappmp.pdf 64 Yves Poirer, Feb, (1995) Production of Polyhydroxyalkanoates, a family of biodegradable plastics and elastomers, in bacteria and plants, Biotechnology, vol13 65 Martin and Williams, App lications of PHAs in Medicine and Pharmacy(2007) retrieved from http://www.tepha.com/media/phaappmp.pdf 66 Yves Poirer, Feb, (1995) Production of Polyhydroxyalkanoates, a family of biodegradable plastics and elastomers, in bacteria and plants, Biotechnology, vol13 67 Kishore, G.M, (1993) Genetic engineeri ng of commercially useful biosynthetic pathways in transgenic plants. Curr-Opin-Biotechnol 68 Poirier, Y. Dennis (1992) Polyhydroxybutyrate, a biodegradable thermoplastic, produced in transgenic plants. Science 256: 520-553

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104 69 Yves Poirer, Feb, (1995) Production of Polyhydroxyalkanoates, a family of biodegradable plastics and elastomers, in bacteria and plants, Biotechnology, vol13 70 Poirier, Y. (1992) Polyhydroxybutyrate, a biodegradable thermoplastic, produced in transgenic plants. Science 256: 520-553 71 Kishore, G.M, (1993) Genetic engineeri ng of commercially useful biosynthetic pathways in transgenic plants. Curr-Opin-Biotechnol 72 Nawrath, C, Poirier, Y. (1994) Plastid targeting of the enzymes required for the production of P3HB in higher plants. Curr-Opin-Biotechnol 73 Yves Poirer, Biotechnology, vol13 Feb, 1995, Production of Polyhydr oxyalkanoates, a family of biodegradable plastics and elastomers, in bacteria and plants. 74 Combined production of PHA biobased polymers and biomass energy, www.metabnolix.org 74 http://www.metabolix.com/biotechnol ogy%20foundation/metabolicengineering.html Diagrams, Tables and Figure Figure 1.1 Stevens, E.S. (2002) Green Plastics Princeton, NJ: Princeton University Press. Figure 1.3McMichael Kirk, (2007) from http://chemistry2.csudh.edu/rpendarvis/Polymer.html Figure 1.4McMichael Kirk, (2007) from http://chemistry2.csudh.edu/rpendarvis/Polymer.html Figure 1.5 Wikipedia.com (2007) from http://en.wikipedia.org/wiki/Extrusion Figure 1.6Chemical Engineers Resource page (2002) from http://www.cheresources.com/injectionzz.shtml Figure 1.7TICONA engineering polymers (2007) from http://www.ticona.com/index/tech/p rocessing/compression_molding.htm

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105 Figure 1.8 Wikipedia.com (2007) from www.wikipedia.com/blowmolding Figure 1.9Offshore Solutions (2007) from http://www.offshoresolutions.com/products/plastic/compressionAndTransferMolding.htm Figure 2.1 McMichael Kirk, (2007) from http://chemistry2.csudh.edu/rpendarvis/Polymer.html Figure 2.2 Encyclopedia Britannica online, (2007 ), Major industri al polymers from www.britannica.com/expoxies Figure 2.3 Wikipedia.com (2007) from www.wikipedia.com/polyethylene Figure 2.4 Wikipedia.com (2007) from www.wikipedia.com/ polyvinylchloride www.kcpc.usyd.edu.au/.../ CelluloseandStarch1.gif Figure 4.1 Chemsoc.org (2007), Structure of cellulose, from http://www.chemsoc.org/ExemplarChem/ent ries/2004/keele_bridge wood/cellulose.gif Figure 4.2Key Center for Poly mer Colloids, retrieved from www.kcpc.usyd.edu.au/.../Ce lluloseandStarch1.gif Figure 4.4Production of polyhydroxyalkanoates, a family of biodegradable plastics and elastomer, in bacteria and plants, Yves Poirer, Biotechnology, volume 13, page 143 Figure 5.1wikipe dia.com (2007) www.wikipedia.com/guttapercha Table 5.1 Biobasics (2006) from http://www.biobasics.gc.ca/english/View.asp?x=790 Figure 5.2 wikipedia.com from http://en.wikipedia.org/wiki/Cellulose_nitrate Table 5.2Biobasics (2006) from http://www.biobasics.gc.ca/english/View.asp?x=790 Figure 5.3NFSA (2007) from www.nfsa.afc.gov.au/Images.nsf/Images/aceta teformula/$File/acetateformula.gif Figure 5.4 wikipedi a.com (2007) from http://en.wikipedia.org/wiki/PLA Figure 5.5Wikipedia.com (2007) from http://en.wikipedia.or g/wiki/Triglyceride

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106 Figure 5.6 Wikipedia.com (2007) from www.wikipedia.com/polycaprolactone, www.wikipedia.com/polyglycolicacid, www.wikipedia.com/polyethyleneoxide www.wikipedia.com/polyviylalcohol. Table 6.1Martin and Williams, Applications of PHAs in Medicine and Pharmacy(2007) retrieved from http://www.tepha.com/media/phaappmp.pdf Table 6.2 Martin and Williams, (2000) Medi cal Applications of poly-4-hydroxybutyate: a strong flexible absorbable material, Biochemical Engineering Journal Figure 7.12 Poirier, Y., Nawrath, C. and Somerville, C. 1995. Production of polyhydroxyalkanoates, a family of biodegradable plastics and elastomers, in bacteria and plants. Bio/Technology 13: 142-149 Figure 7.13 Lindsey, Keith. 1998. Transgenic Plant Research. Harwood Academic Publishers. Amsterdam, The Netherlands. 201-219 pp Figure 7.14 Lindsey, Keith. 1998. Transgenic Plant Research. Harwood Academic Publishers. Amsterdam, The Netherlands. 201-219 pp


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