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Steam Hydrolysis and Anaerobic Digestion of Biodegradable (polylactic Acid) Packaging Waste

Permanent Link: http://ufdc.ufl.edu/UFE0025168/00001

Material Information

Title: Steam Hydrolysis and Anaerobic Digestion of Biodegradable (polylactic Acid) Packaging Waste
Physical Description: 1 online resource (66 p.)
Language: english
Creator: Moreira, Cesar
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: anaerobic, commercial, digestion, hydrolysis, pla
Agricultural and Biological Engineering -- Dissertations, Academic -- UF
Genre: Agricultural and Biological Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Biodegradable plastic is now being used for packaging purposes to avoid the environmental burden of oil based polymers. However, after usage the biodegradable polymer waste when disposed takes longer to degrade than typical organic wastes. The same mechanical characteristics that make this plastic appealing to use make it undesirable in composting operations. Relatively high temperature industrial composting conditions are required to treat this waste. Most municipalities lack such facilities. Therefore, the majority of biopolymer wastes are sent to landfills, which is contrary to the purpose of using biodegradable polymers. Research has suggested pretreatment as a solution for accelerating degradation of biopolymers. Among proposed pretreatment, hydrolysis has been suggested as the most promising. In this research kinetics of hydrolysis of polylactic acid at temperatures above and below the melting point of the polymer, and mass loading of PLA during hydrolysis was studied to explore the extent of degradation of the material on reaction. In all cases studied, loss in mass and degradation of polymer was observed. It was most noticeable when 2.5 grams and 7.5 grams of sample was exposed for 120 minutes at 160 masculine ordinalC. At the end of the treatment no solids were present, and the molecular weight average (MW) reduced to 900 and 1217 respectively. Previous work done in our laboratory discovered that it was possible to digest PLA in a thermophilic anaerobic digester and this would serve to eliminate waste while producing methane. In this work benefits of anaerobic digestion of PLA using an adapted microbial flora were studied. Results of this work suggest that PLA can be directly digested anaerobically without need for hydrolysis pretreatment. The digestion of hydrolyzed PLA was very fast, and depending on the time of exposure the lag time was between 0.64 and 3.5 days, when compared to a lag time of 25 days when using raw PLA. Anaerobic digestion yielded 94 -98% of theoretical methane yield, indicating almost complete biogasification of the material.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, 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.
Statement of Responsibility: by Cesar Moreira.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Welt, Bruce A.
Local: Co-adviser: Pullammanappallil, Pratap C.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025168:00001

Permanent Link: http://ufdc.ufl.edu/UFE0025168/00001

Material Information

Title: Steam Hydrolysis and Anaerobic Digestion of Biodegradable (polylactic Acid) Packaging Waste
Physical Description: 1 online resource (66 p.)
Language: english
Creator: Moreira, Cesar
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: anaerobic, commercial, digestion, hydrolysis, pla
Agricultural and Biological Engineering -- Dissertations, Academic -- UF
Genre: Agricultural and Biological Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Biodegradable plastic is now being used for packaging purposes to avoid the environmental burden of oil based polymers. However, after usage the biodegradable polymer waste when disposed takes longer to degrade than typical organic wastes. The same mechanical characteristics that make this plastic appealing to use make it undesirable in composting operations. Relatively high temperature industrial composting conditions are required to treat this waste. Most municipalities lack such facilities. Therefore, the majority of biopolymer wastes are sent to landfills, which is contrary to the purpose of using biodegradable polymers. Research has suggested pretreatment as a solution for accelerating degradation of biopolymers. Among proposed pretreatment, hydrolysis has been suggested as the most promising. In this research kinetics of hydrolysis of polylactic acid at temperatures above and below the melting point of the polymer, and mass loading of PLA during hydrolysis was studied to explore the extent of degradation of the material on reaction. In all cases studied, loss in mass and degradation of polymer was observed. It was most noticeable when 2.5 grams and 7.5 grams of sample was exposed for 120 minutes at 160 masculine ordinalC. At the end of the treatment no solids were present, and the molecular weight average (MW) reduced to 900 and 1217 respectively. Previous work done in our laboratory discovered that it was possible to digest PLA in a thermophilic anaerobic digester and this would serve to eliminate waste while producing methane. In this work benefits of anaerobic digestion of PLA using an adapted microbial flora were studied. Results of this work suggest that PLA can be directly digested anaerobically without need for hydrolysis pretreatment. The digestion of hydrolyzed PLA was very fast, and depending on the time of exposure the lag time was between 0.64 and 3.5 days, when compared to a lag time of 25 days when using raw PLA. Anaerobic digestion yielded 94 -98% of theoretical methane yield, indicating almost complete biogasification of the material.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, 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.
Statement of Responsibility: by Cesar Moreira.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Welt, Bruce A.
Local: Co-adviser: Pullammanappallil, Pratap C.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025168:00001


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1 STEAM HYDROLYSIS AND ANAEROBIC DIGESTION OF BIODEGRADABLE (POLYLACTIC ACID) PACKAGING WASTE By CESAR M. MOREIRA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Cesar M. Moreira

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3 To God who gave to me a wonderful family and friends always ready to give support, moral and advice

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4 ACKNOWLEDGMENTS I would like to thank my professors, adv isors, staff, and classmates in the ABE department for their support, understanding and trust along my academic career. I appreciate Dr. Bruce Welt (chair) and Dr. Pratap Pullammannappall il (cochair) support and advice; also, to my lab-mates Abhay Koppar and Jaime V. Chavez-Leon for their guidance and help. In a special note I woul d to thank The Butler Polymer Laboratory especially Mr. Laurent Mialon (Chemistry Department), and Dr. Adegbola Adesogan Laboratory (Animal Science) in this re search for their invaluable help.

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5 TABLE OF CONTENTS page ACKNOWLEDG MENTS .................................................................................................. 4LIST OF TABLES ............................................................................................................ 7LIST OF FIGURES .......................................................................................................... 8ABSTRACT ..................................................................................................................... 9 CHA PTER 1 INTRODUC TION .................................................................................................... 112 HYDROLYSIS AND BIODEGRADATION OF POLYLACTIC ACID (PLA) .............. 143 POLYLACTIC ACID HYDROL YSIS ........................................................................ 20Introduc tion ............................................................................................................. 20Background ............................................................................................................. 20Material and Methods ............................................................................................. 22Feedsto ck ......................................................................................................... 22Feedstock Pr eparation ..................................................................................... 22Hydrolysis Pr otocol ........................................................................................... 22Analysis .................................................................................................................. 23Results .................................................................................................................... 24Discuss ion .............................................................................................................. 26Molecular Weight Degradation ......................................................................... 26Lactic Acid Formati on ....................................................................................... 27Effect of PLA Concent ration on Hy drolysis ....................................................... 28Effect of Temperat ure on Hydr olysis ................................................................ 28Conclusi ons ............................................................................................................ 294 ANAEROBIC DIGESTION OF HYDROLYZED AND NON-HYDROLYZED POLYLACTIC ACID ................................................................................................ 42Introduc tion ............................................................................................................. 42Background ............................................................................................................. 44Material and Methods ............................................................................................. 48Feedsto ck ......................................................................................................... 48Anaerobic Digestion Protoc ol ........................................................................... 48Mixed microbial flora (inocolum) pr eparation ............................................. 48Feedstock pr eparation ............................................................................... 49BMP prepar ation ........................................................................................ 49Analysis .................................................................................................................. 49Results .................................................................................................................... 50

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6 Discuss ion .............................................................................................................. 50Future Work ............................................................................................................ 54LIST OF RE FERENCES ............................................................................................... 61BIOGRAPHICAL SKETCH ............................................................................................ 66

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7 LIST OF TABLES Table page 3-1 Molecular weight degradat ion of PLA at 121C .................................................. 37 3-2 Molecular weight degradation of PLA 160C ...................................................... 38 3-3 Molecular weight PLA degr adation constant k and Ea ....................................... 39 3-4 Lactic acid first order rate c onstant s ................................................................... 39 3-5 Primary transition temperatur es of selected PLA copoly mersa ........................... 40 3-6 Effect of processing conditions on mechanic al properties of PLA copolymersa .. 41 4-1 Hydrolyzed se lected samples ............................................................................. 58 4-2 Lactic acid concentration and percent recovery of hydrolyzed selected samples .............................................................................................................. 58 4-3 Content of BMP bo ttles ...................................................................................... 59 4-4 PLA molecular weig ht after hy drol ysis ............................................................... 59 4-5 Summary of performance of hy dro lyzed and non hy drolyzed PLA ..................... 60

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8 LIST OF FIGURES Figure page 3-1 Cup used for obt aining f eedsto ck ....................................................................... 30 3-2 MW degra dation 121C ...................................................................................... 31 3-3 MW degra dation 160C ...................................................................................... 32 3-4 Variation in lactic acid concentration as a function of time. A) Lactic acid concentration while working at 121 oC. B) Lactic acid concentration while working at 160 oC ............................................................................................... 33 3-5 Linearized plot of rate of change with respect to time A) Model and Experimental data at 121 C B) M odel and Ex perimenta l data at 160C ............ 34 3-6 Determination of Ea fo r MW degradatio n ........................................................... 35 3-7 Lactic acid concentration plotted as first order equation A) 2.5 and 7.5 grams 121 C B) 2.5 and 7. 5 grams 160 C ................................................................... 36 4-1 Mixed microbial flora adapt ation ......................................................................... 55 4-2 PLA anaerobic digestion. A) Hy drolyzed PLA. B) Raw PL A. .............................. 56 4-3 Initial methane production ra te from hydr olyzed PL A. ........................................ 57 4-4 Methane production from hydrolyzed PLA and raw PLA in the first 21 days of anaerobic diges tion. ........................................................................................... 57

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9 Abstract of Thesis Pres ented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Master of Science STEAM HYDROLYSIS AND ANAEROBIC D IGESTION OF POLYLACTIC ACID PACKAGING WASTE By Cesar M. Moreira December 2009 Chair: Bruce A Welt Cochair: Pratap Pullammanappallil Major: Agricultural and Biological Engineering Biodegradable plastic is now being used for packaging purposes to avoid the environmental burden of oil based polymers. However, after usage the biodegradable polymer waste when disposed takes longer to degrade than typical organic wastes. The same mechanical characteristics that make this plastic appealing to use make it undesirable in composting operations. Relatively high temperature industrial composting conditions are required to treat this waste. Mo st municipalities lack such facilities. Therefore, the majori ty of biopolymer wastes are se nt to landfills, which is contrary to the purpose of using biodegradable polymers. Research has suggested pretreatment as a solution for accelerating degradation of biopolymers. Among proposed pretreatment, hydrolysis has been suggested as the most promising. In this research kinetics of hydrolysis of polylactic acid at temperatures above and below the melting point of the polymer, and mass loading of PLA during hydrolysis was studied to explore the extent of degradat ion of the material on reaction.

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10 In all cases studied, loss in mass and degradation of polymer was observed. It was most noticeable when 2. 5 grams and 7.5 grams of sample was exposed for 120 minutes at 160 C. At the end of the tr eatment no solids were present, and the molecular weight average (MW) reduc ed to 900 and 1217 respectively. Previous work done in our laboratory discove red that it was possible to digest PLA in a thermophilic anaerobic digester and this would serve to eliminate waste while producing methane. In this work benefits of anaerobic digestio n of PLA using an adapted microbial flora were studied. Result s of this work suggest that PLA can be directly digested anaerobically without need fo r hydrolysis pretreatment. The digestion of hydrolyzed PLA was very fast, and depending on the time of exposure the lag time was between 0.64 and 3.5 days, when compared to a lag time of 25 days when using raw PLA. Anaerobic digestion yielded 94 -98% of theoretical methane yield, indicating almost complete biogasif ication of the material.

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11 CHAPTER 1 INTRODUCTION Success of plastics is evidenc ed by its use in many types of packaging throughout the world. However, plastic waste is often viewed as an environmental burden. Several authors have defined polyolefin plastics as energy sinks since the material is made from fuel stocks and requires even more energy to c onvert fuel into plastics. The three most important polyolefins used from producti on of petrochemicals are polyethylene, polypropylene and polybutadie ne (Hatch et al., 1981). Traditional sources for olefins production in Europe and in the United States are naphtha, gas oil and liquid gas petroleum. These raw materials must undergo energy intensive transformations to obtain ethylene, propylene, or butadiene. Also, other materials are added to olefins to form more co mplicated structures, such as nitrogen to propylene to obtain acrylonitrile, oxygen to obtain epoxies, etc. (Hatch et al., 1981). A polymer is a macromolecule with large numbers of repeating units. Homopolymers are made from one building block. Copolymers are made from more than one building block produced by t he addition polymerization reactions (Wojciechowski et al. 1986). Polymers are classified depending on type of monomer (polyolefins, polyesters, polyamides, etc.); type of formation reaction (condensation or addition polymerization); type of thermal behavior (thermosetting and t hermoplastics); and type of utilization, (thermosetting, thermoplastics, fibers, engineering plastics, etc.). Polymer data are usually given under the name of the polymer such as polyethylene, polystyrene, or by the type of monomer as acrylic or polyester.

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12 Design of material with specific useful liv e requires choice of specific monomers to obtain copolymer with the desirable hydrophili c characteristics (Le Digabel and Averous, 2006; Yew et al., 2006) In an effort to relieve the real or perceived environmental burden of petroleum based polymers, work is being done to develop bi ologically derived plastics. Polylactic acid (PLA), which is produced from r enewable plant resources, has recently experienced increased utilization as an alter native to petro-derived polymers in order to reduce their impact on t he environment (Tsuji, 2008). PLA belongs to the family of alip hatic polyesters commonly made from -hydroxy acids, which includes polyglycolic acid or polymandelic acid, and are considered biodegradable and compostable. PLA is a polyester polymer produced by t he condensation of lactic acid that is derived from microbial fermentation of r enewable agriculture resources, such as glucose from corn, sucrose from cane sugar lactose from cheese whey, and cellulose from waste papers (Ho et al., 1999). PLA is a thermoplastic, with hi gh strength and high modulus, which makes it useful for the i ndustrial the industrial packaging or the biocompatible/bioabsorbable medical device market. It is one of the few polymers in which the stereochemical structure can easily be modified by polymerizing a controlled mixture of the Lor Dis omers to yield high molecular weight amorphous or crystalline poly mers that can come in contact with food and are generally recognized as safe (GRAS)(Garlotta, 2001). LPolylactic acid (PLLA) has also been attr acting much interest as an alternative to commercial polymers such as pol yethylene, polypropylene, polyethylene

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13 terephthalate, and polystyrene. PLLA is also a superior material for feedstock recycling into L-lactic acid by hydrolysis and into L, L-Lactide by pyrolysis (Mohd-Adnan et al., 2008). Hydrolysis is a process by which polym ers undergo chemical degradation by being split by addition of water; the polymer mu st contain hydrolysable covalent bonds such as ester, ether, anhydride, amide, carbamide (urea), ester amide (urethane) etc. Rate and extent of hydrolysis (degr adation) depends on parameter such as water activity, temperature, pH and time (Le Digabel and Averous, 2006) PLA degradation occurs in the presence of water provoking a hydrolysis of the ester bonds (Lucas et al, 2008). The rate of degradation depends on size and shape of the article, the isomer ratio, and temperature hy drolysis (Garlotta, 2001). Today many cups and containers manufactu red with PLA are going straight to landfills because most munici palities do not possess expertise or equipment to handle PLA. Nevertheless, this biodegradable plastic does not degrade quickly in landfills (Tokiwa et al., 2004). This research objective is study of the degradation of PLA due to hydrolysis and possible recovery of valuable by-products.

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14 CHAPTER 2 HYDROLYSIS AND BIODEGRADATION OF POLYLACTIC ACID (PLA) Currently, PLA is used for medical applicat ions including wound c losure, prosthetic implants, and drug release (Tokiwa and Calabi a, 2006); as controlled-release devices for herbicides and pesticides; and as a plant growth enhancer (de Jong et al., 2001). Developing applications for PLA include degr adable plastics (cast films, blown films, and rigid containers), fibers and nonwovens, and paperboard coatings. PLAdegradable plastics have a projected U.S. market of 2.5 to 3.4 million tons/year (sales volume, $3.1 to 4.4 billion/year) and are expected to compete with hydrocarbon-based thermoplastics, such as polystyrene, pol ypropylene, polyethylene, and polyethyleneterephthalate, on a cost and perfo rmance basis (Ho et al., 1999). PLA has high mechanical strength, thermal plasticity, fabricabilit y, biodegradability, and biocompatibility. It has been proposed as a renewable, degradable plastic for uses in service ware, grocery, waste and co mposting bags, mulch films and controlled release matrices for fertilizers, pesticides and herbicides. Generally, PLA polymers are made into us eful items using thermal processes, such as injection molding and extrusion. Therefore, its rheological properties, especially its shear viscosity are import ant to processes such as film blowing, paper coating, injection molding, sheet forming and fiber spinning. In order for PLA to be processed on lar ge-scale production lines in applications such as injection molding, blow molding, thermoforming, and extrusion, the polymer must possess adequate thermal stability to pr event degradation and maintain molecular weight and properties.

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15 Pure PLA undergoes thermal degradation at temperatures above 200 C (392 F) by hydrolysis, lactide reformation, oxidative main chain scission, and interor intramolecular transesterification reactions (Jamshidi et al., 1988). The most widely used method for improving PLA processabili ty is based on melting point depression by random incorporation of sma ll amounts of lactide enantiomers of opposite configuration into the polymer (i.e. adding a small amount of D-lactide to the L-lactide to obtain PDLLA). Unfortunately, the me lting point depression is accompanied by a significant decrease in crystallinity and crystallization ra tes (Garlotta, 2001). PLA can be easily degraded by enzymatic or alkali hydrolysis in com post, but its rate of degradation in soil is not high (Ohkita, 2006). Also, PLA plasti cs are sensitive to moisture and heat, which limits applications for the pl astic (Ho et al., 1999). Polymeric materials that are exposed to outdoor conditions (i.e. weather, ageing and burying) undergo degradation from mechani cal actions/interactions and light, thermal and chemical reactions (Helbi ng et al., 2006; Ipekoglu et al., 2007). Thermal degradation of thermoplastic polymer s occurs at the melting temperature when the polymer is transformed from solid to liquid (159 178 C for LPLA depending on molecular weight). Biodegradable polymers such as L-PLA are semicrystalli ne polymers, they posses amorphous and crystalline regions Structural changes take place at their glass transition temperature (Tg) (i.e. 50 C for L-PLA), the m obility and volume of polymeric chains are modified. Above Tg (rubbery state), disorganization of chains facilitate chemical and biological reactions. Below Tg (glassy state), formation of spherulites may take place, generating inter-spher ulitic cracks and brittleness.

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16 Hydrolysis is another way by which pol ymers undergo chemical degradation. PLA hydrolysis occurs in the presence of water provoking a hydrolysis of ester bonds. (Lucas et al., 2008) Hydrolytic degradation of t he polymer matrix is affected by the amount of crystallinity in the samples. It has been s hown that highly crystalline PLA will take months, sometimes year s, to hydrolyze fully to lact ic acid, whereas an amorphous sample degrades in weeks. This is due t he impermeability of cr ystalline regions. Pure poly(D-lactide) or poly(L-lactide) has an equilibrium crystalline melting point of 207 C, but typical melti ng points are in the 170 C 180 C range. This is due to small and imperfect crystallites, slight racemization, and impurities (K haras et al., 1994; Kricheldorf et al., 1996). In PLA hydrolytic degradati on, degradation varies depending on time, temperature, molecular weight, impurities, and catalyst concentration. Cata lyst and oligomers decrease degradation tem perature and increase degradation rate of PLA. In addition catalysts cause viscosity and rheological changes (Garlotta, 2001). PLA Hydrolysis is catalyzed by both acid and base. (Gopferich et al., 1996) Hydrolytic degradation of ma ssive amorphous poly (DL-lactic acid) devices was shown to proceed heterogeneously, proceeding faster inside than at the surface (Vert et al., 1994). In the interior there is a la rger contribution of auto-catalysis. Initially, hydrolysis of ester bonds proceeds homogenously trough the ma trix. During degradation, two factors are of importance. First, degradation causes an increase in the number of carboxylic acid chain ends, which are known to auto-ca talyze ester hydrolysis. Second, only

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17 oligomers, which are solubl e in the surrounding aqueous medium, can escape from the matrix. As aging proceeds, soluble oligomers, which are close to the surface, can leach out before they fully degrade, whereas those located in the core of the matrix remain entrapped. This yields a low pH in the core which, in turn, results in accelerated degradation rates (Vert et al., 1994). Degradation of semi-crystalline PLLA invo lves even more complex processes. It was reported by Fischer et al 1973 that the hydrolytic degr adation occurs in two stages. In the first stage, water di ffuses into the amorphous regions resulting in random hydrolytic scission of ester bonds. The degr ee of crystallinity can even increase as degradation proceeds. The second stage starts when most of the amorphous regions have been degraded. Hydrolytic attack then pr ogresses from edges towards the center of crystalline domains. A retardation in degradation has been observed during degradation of intrinsically am orphous poly (DL-LA) by the formation of a crystalline phase of an oligomeric stereocomplex as an intermediate. This intermediate setereocomplex is highly resistant to hydrolysis (S.J. de Jong et al., 2001). Biodegradation of polymeric materials incl udes several steps and the process can stop at each stage (Pelmont., 1995). The combined action of microbial co mmunities, other decomposer organisms or/and abiotic factors fragment the biodegradable materials in to tiny fractions. This step is called biodeterioration (Eggins and Oxley, 2001; Walsh, 2001). Microorganisms secrete catalytic agents (i .e. enzymes and free radicals) able to cleave polymeric molecules progressively reducing molecular weight. This process generates oligomers, dimmers and monomers. This step is called depolymerisation. Some molecules are recognized by receptors of microbial cells and can move across membranes. Other molecules stay in the extracellular surroundings and can be the object of different modifications.

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18 In the cytoplasm, trans ported molecules integrate in to microbial metabolism to produce energy, new biomass, storage vesicles and numerous primary and secondary metabolites. This step is called assimilation. Concomitantly, some simple and comp lex metabolites may be excreted and reach the extracellular surroundings (e.g. organic acids, aldehydes, terpens, antibiotics, etc.) Simple molecules such as CO2, N2, CH4, H2O and different salts from intracellular metabolites that are completely oxidized are released in the environment. This stage is called mineralization. The term biodegradation indicates the predominance of biological activity. However, in nature, biotic and abiotic fa ctors act synergically to decompose organic matter. Several studies about biodegradation of some polyme rs show that the abiotic degradation precedes microbial assimilation Consequently, the abiotic degradation must not be neglected (Lucas et al., 2008). Several studies have shown that certai n proteases including proteinase K, pronase and bromelain have bee n found to increase the rate of degradation of PLA Williams (1981) was the first to report biodegr adation of PLA by Proteinase K, a fungal serine protease of Tritirachium album. PLA can be degraded as well by microbes ; however, PLA degraders appear to be scarce in the environment. A burial test indicated that PLA was not readily degraded when samples were buried under soil for 20 months (Tokiwa et al., 2004). Pranamuda et al., (1997) found that PL A degrading organisms are sparsely distributed in soil environments and found only one, an actinomycete Amycolatopsis sp. that degraded PLA in culture at 30 C. Hakka rainen et al., (2002) fo und that PLA films were degraded to a powder after 5 wee ks in a mixed culture of compost microorganisms at 30C whereas t he film in abiotic medium looked intact. They also found PLA molecular weights, especially number average mo lecular weight (Mn) were reduced to a greater extent in the biot ic medium, probably due to cleavage near the

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19 chain ends. Other authors claim that initial degradation is due to abiotic hydrolysis only followed by biotic assimilation of breakdown products. PLA is completely mineralized to CO2, water and a small amount of biomass afte r 4-6 weeks in compost (Shogren et al., 2002) (in composting aerobic conditions prevails.) Under anaerobic conditions, organic matte r usually degrades in four stages: a) hydrolysis, b) acidogenesis, c) ac etogenesis, and d) methanogenesis. During hydrolysis molecules split and become smalle r and soluble resulting in conversion of carbohydrates, fats and proteins into sugars, fatty acid and amino acids. This chemical reaction requires water, and is aided by temperature and enzymes. Later, during acidogenesis, simpler compounds undergo fe rmentation carried out by acidogenic bacteria, and produce volatile fatty acids, hydrogen and carbon di oxide. Later, during acetogenesis, acetic acid, hydrogen, and carbon dioxide are produced. During methanogenesis, the final produc ts of the anaerobic digesti on are obtained. These are methane and carbon dioxide. Little information was available for the anaerobic degradation of PLA, and most of the information available pertains to com posting. PLA can be degraded in a composting environment where it is hydrolyzed into smaller molecules (oligomers, dimmers and monomers) after 45-60 days at 5060 C (Urayama et al., 2002). Anaerobic digestion could be a valid alternative for PLA degradation. The anaerobic digestion will lead to a complete mi neralization of PLA which is much better than the non-degradation happening in land fills and the energy consumption required performing high temperature composting. T herefore, studies were done to determine feasibility of anaerobic digestion of PLA in order to produce methane.

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20 CHAPTER 3 POLYLACTIC ACID HYDROLYSIS Introduction Characteris tics that make PLA a desirabl e material (i.e. excellent mechanical properties, stability and durability) make it resistant to degra dation. As a result, PLA has a low biodegradability rate compared to composted organic waste, this prevents commercial compost operators from receiving it. Several pretreatments hav e been proposed as a solution with hydrolysis being most effective (Vargas et. al. 2007). Hydr olysis may offer an opportunity for monomer recovery. The chapter describes effects of te mperature and moisture on PLA. Two temperatures and three ratios of PLA-water were studied to better understand hydrolytic degradation of PLA. Background Polylactic acid (PLA) is a bio-degradable thermoplastic polymer that is beginning to be produced on large scale from fermentation of corn to lactic acid and subs equent polymerization (Shoegren et. al 2001). Biodegradable polyesters, such as PLA are under investigation for biomedical applications including ort hopedic fixture materials, degr adable sutures, absorbable fibers and pharmaceutical applications such as controlled-release devices (de Jong et. al. 2001). PLA has been used as a matrix for the controlled-release of drugs and as scaffolds on which living tissue can regenerate (Khang et al., 2003). Also, PLA has been proposed as a renewable degradable plastic for use in service-ware, grocery,

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21 waste and composting bags, mulch films, and c ontrolled release matrices for fertilizers, pesticides and herbicides (Qi Fang et. al. 1999). The percentage of poly(L-lactide) (PLLA) and poly D-lactide (PDLA) in polymer blends affect crystal structur e, melting point, and glass tr ansition of PLA (Okihara et al.,1991; Garlotta 2002). A 50/50 PLLA/PDL A in polymer blend can have a different crystal structure from that of pure PLLA or PDLA (Sasaki, 2003). The 50/50 blend can form a stereo complex, which is a co mplex between PLLA and PDLA. The stereo complex structure of the 50/50 PLLA/PDLA blend has a gl ass transition temperature of 65-72 C (Tsuji, 2002;2005) and a melting point of 220-230 C, which are higher than those of pure PLLA and pure PDLA (Tsuji, 2002; Sarasua, 2005). Several methods have been proposed for PL A degradation. However, Vargas et al. (2009) compared several degradation methods including gamma irradiation, electron beam irradiation and steam treatment (hydro lysis). Results of this study showed hydrolysis as the most effective pretreatment to degrade polylactic acid. Hydrolytic degradation proceeds either at the surface (homogeneous) or within the bulk material (heterogeneous) and is controlled by a wide variety of compositional and property variables such as ma trix morphology, chain orient ation, chemical composition and stereochemical structure, sequence distribut ion, molecular weight and distribution, presence of residual monomers, oligomers and other low molecular weight products, size and shape of the specimen, oxygen, microorganisms, enzymes, pH and temperature (Hakkarainen, 2002). Degradation occurs in stages, the first bei ng diffusion of water into the material, hydrolysis of ester bonds and lowering of mo lecular weight followed by intracellular

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22 uptake of lactic acid oligomers, and catabol ism. Rates of hydrolysis increase with water content and temperature and are catalyzed by free carboxyl groups of the hydrolyzed PLA ends. Hydrolysis is actually faster in the interior of a thick sample since carboxylic acid concentrations are higher than at the exterior due to leaching of the acidic PLA oligomers into the surr oundings aqueous medium. In abiotic aqueous environments degradatio n proceeds through hydrolysis of the ester bond, this reduces molecular weight polymer to intermediate degradation products (insoluble and soluble oligom ers) and, finally, lactic acid is formed as the ultimate degradation product of abiotic hy drolysis (Shogren et. al. 2003). Material and Methods Feedstock PLA waste was created using commercial t hermoformed cups (Fabri-Kal Inc ., Kalamazoo, MI) obtained from TR EEO Center at the University of Florida (Figure 3-1). Feedstock Preparation 2 packages of 50 cups each were ground in a hammer mill. After grinding pieces were cut manually until homogeneous pieces of approximately 1 by 0.25 were obtained. Hydrolysis Protocol Step 1: three concentrations of PLA were tried, so 2.5 gr ams, 7.5 grams and 30 grams in 30 grams of water. Step 2: to perform hydrolysis a Mathis BFA-24 Beaker Dryer with PLC Univision (Werner Mathis USA Inc ., Concord,NC) was used. Step 3: Waste samples were deposited in the Mathis BFA-24 200 mL vials (working pressure 4 bar) with 30 grams of D.I. water. Step 4: Vials were purged for 2 minut es using nitrogen gas, and then tightly closed.

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23 Step 5: For each concentration duplicates were made. Step 6: Vials were loaded into the machine. Step 7: Mathis dyer was programmed to stay at 121 C, and the waste was treated for 120, 240, 360, 480 and 720 minutes. Step 8: Vials were allowed to cool down. Step 9: Once vials were at room temperature, 15 mL of liquid sample were taken from each vial and filtered us ing Whatman syringe 0.45 m filters. The rest of the vial including solids was placed in an oven at 95 C for 48 hours to drive off water. Step 10: 2 mL from the filtered liquid was taken from the vials containing the liquid sample, and placed into a clear borosilicate glass screw-neck 12x32 mm numbered vials (sample used for lactic acid determination). Step 11: Solids were taken from the ov en and 10 mg sample were weighed and placed into 10 mL screw-neck pyrex vials; 10 mL of tetrahydrofuran (THF) HPLC grade was added to each vial. The vial wa s agitated and warmed for complete dissolution of the sample (sample used for molecular weight determination). Step 12: remaining solids were weighed and then stored. Steps 1 to 6 were repeated for different concentrations (2.5, 7.5 and 30 grams) Step 13: the Mathis dyer was then program med to remain at 160 C and the waste was treated for 30, 60, 90, and 120 minutes. Steps 8 to 11 were repeat ed for each concentration. Analysis A Hitachi UV reverse phase HPLC was used to determine lactic acid. The mobile phase used was a solution of sulfuric acid Chromatograms of samples treated at 121 C for more than 360 minutes needed to be dilut ed. For this analysis at time 0, lactic acid concentration was zero. A Waters GPCV2000 gel permeation ch romatograph was used to determine molecular weight of the different sample s. tetrahydrofuran (THF) was used as the mobile phase in this case. Initially a sample of raw PLA was analy zed.

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24 Results In all cases reduction of molecular weight and growth in lactic acid concentration was observed (Figure 3-2, 3-3, 3-4). Fi gure 3-2 summarizes degradation of polylactic acid at 121 C for 2.5 grams and 7.5 grams of PLA loading, respectively. The plot is an average of molecular weights for two runs. T he molecular weight at the start of each PLA loading was determined to be 1.21 x 105 grams. For the sample with 2.5 grams, molecular weight decrease rapidly to 2.8 x 104 grams in 120 minutes and continued to decrease further to 1.09 x 104 grams at 360 minutes. At the end of the run (720 minutes) molecular weight was 5.57 x 103 grams. Similar observations were noted for PLA loading of 7.5 grams. After 720 mi nutes the molecular weight was 4.7 x 103 for 7.5 grams of PLA loading (Table 3-1). Figure 3-2 and 3-3 summari zes degradation of polylactic acid at 160C for samples made with 2.5 grams and 7.5 grams PL A, respectively. The molecular weight at the start of each PLA loadi ng was determined to be 1.21 x 105 grams. For samples with 2.5 grams PLA the molecular we ight decreased rapidly to 7.85 x 103 grams in 30 minutes and continued to decrease to 5.92 x 103grams at 45 minutes. At the end of the run (120 minutes) molecular weight dropped to 9.27 x 102 grams. A similar observation was noted for PLA samples of 7.5 grams. After 120 minutes, molecular weight was 7.24 x 102grams (Table 3-2). Figure 3-5A summarizes the increase in lact ic acid concentration at 121 C for 2.5 grams and 7.5 grams of PL A, respectively. The plot is an average concentration of lactic acid for two runs. Initial lactic acid c oncentration was determined to be 0. Samples with 2.5 grams PLA lactic acid conc entration increased to 1.30 x 10-3 M in 120 minutes and continued to increase further to 1.02 x 10-2 M at 240 minutes. At t he end of the run (720

PAGE 25

25 minutes) the lactic acid increases to 3.2 x 10-1 M. A similar observation was noted for 7.5 grams PLA samples. After 720 minutes t he lactic acid concentrations was 1.11 M. Figure 3-5B shows lactic acid trends at 160 C for 2.5 grams and 7.5 grams of PLA loading respectively. The plot is an average of lactic acid production for two runs. Lactic acid concentration at the start of each PLA loading was determined to be 0 M. For samples with 2.5 grams PLA lactic acid concentration increase to 1.1 x 10-2 M in 30 minutes and continued to increase to 7.0 x 10-2 M and 1.5 x 10-1 M. At the end of the run (120 minutes) lactic acid concentration was 3.51 x 10-1 M. A similar observation was noted for PLA loadings of 7.5 grams. After 120 minutes the lactic acid concentrations was 4.43 x 10-1. A first order model was proposed to describe the kinetics of degradation. Equation (3-1) is a general expression fo r first order kinetics. (3-1) where A: concentration of the reactant (mol) k: rate constant (min-1) n: order of the reaction t: time (min) This expression was linearized to determine constant k (slope). Figure 3-7shows a normalized plot of rate of change of mo lecular weight with respect to time. (3-2) where Ao: initial concentration of the reactant (mol) k: rate constant (min-1) n: order of the reaction t: time (min)

PAGE 26

26 Table 3-3. shows constants, k and n for 121 and 160C. Fig 3-6 shows the calculation of energy of activation (Ea) and table 3-3, exhibits the summary of the obtained values. To understand kinetics of lactic acid pr oduction it was proposed to determine the order of the reaction. The equation below is a general expr ession to determine first order reaction. dP/dt = k[A] (3-3) where A: concentration of the reactant (mol) P: concentration of the product (mmol/L) k: rate constant (min-1) t: time (min) This expression was linearized to determi ned constant k (inter cept). Figure 3-7 shows a semi-log plot of rate of change of molecular weight with respect to time. Ln [P] = [A] e-kt (3-4) where A: concentration of the reactant (mol) P: initial concentration of lactic acid (mmol/L) k: rate constant (min-1) t: time (min) Table 3-4 shows constants, k and n for 121 and 160C. Discussion Molecular Weight Degradation The reduction in molec ular weight happens with simultaneous loss of PLA mass. Since the matter can not be destroyed, the lo ss in PLA mass suggests the formation of another product as effect of degradation. Literature suggests that moisture br eaks the ester bonds of PLA producing a reduction in chain size. Degradation of polylactic acid shows an exponential trend (Fig 3-2 and 3-3). Therefore, first order kinetics were applied. Fo r this experiment molecular

PAGE 27

27 weight average, Mw, was used. Many aut hors have been experimenting with kinetics models for PLA hydrolytic degradation; however, temperat ure, concentration, heat exposures vary, which results in different kinetics rate constants (k), and energy of activation (Ea). Activation energy (Ea) was estimated us ing values of k at 121 and 160 C. Ea calculated was 7.4 x 104 J/mol (Table 3-3) and the k values were 7.7 x 10-3 and 5.9 x 10-2 min-1 respectively. Tsuji et al. (2004) reported an Ea value of 7.52 x 104 J/mol when working with a range of temperatures of 37-97C. Mohd-Adnan et al. 2008 reported different values for autocatalytic random hydrolysis being considered as the main reaction mechanism. This study suggests that employment of unsuitable reactions mechanisms to analyze kinetics can produce large deviations in Ea value. k values at 100 C were 8.4 x 10-5 and 7.2 x 10-4 s-1 at 130 C resulting in an Ea of 8.72 x 104 J/mol Lactic Acid Formation Production of lactic acid happened as a re sult of PLA degradation. Lactic acid production did not follow the same trend of PL A degradation. Most of the degradation of PLA happened in the first 120 minutes w hen the heat exposure was 121 C and 30 minutes when the heat exposition was 160 C. Ho wever, the concentration of lactic acid at 120 minutes and 30 minutes when using a heat exposure of 121 C and 160 C was low. Production of lactic acid shows an exponential trend (Figure 3-4). First order kinetics fits well to data after initial delay. Initial composition of the waste is unknown, but for effect of calculations it was assumed PLA after hydrolysis yielded only lactic acid. Knowing the exact amount of PLA and water added and using PLA density of 1.25 g/mL initial Molar solution were calculated, and then compared with the results of HPLC analysis. At 121 C the maximum recovery (7.5 grams and 720 minutes) was about

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28 47% and at 160 C the maximum recovery (7.5 gr ams and 120 minutes) was about 20%. An increase in acetic acid concentration was observed when working at 160 C. Hydrolysis of optically active PLLA in the melt may cause racemization and decomposition of lactic acids due to high te mperature Tsuji et al (2003). PLA is not 100% lactic acid Garlotta et al. (2001) in his literature review of polylactic acid shows several compositions for starch-PLA plasticizers; overall the standard lactic acid concentration in PLA ranges from 60-75%. Effect of PLA Concentration on Hydrolysis Results suggest that the amount of PLA tr eated affected the hydrolysis. In this particular experiment, a better fi t to a first order kinetics was observed for samples with 7.5 grams of PLA; this phenomenon repeats with both heat exposures (121 C and 160 C). Zhang et al., 1994 conducted studies on polylactic acid degradation and suggested that polymer degradation rate is determined by polymer reactivity with water and accessib ility of ester groups to wate r and catalyst (carboxylic end groups). Effect of Temperature on Hydrolysis It can be seen from table 3-1 and 3-2 the molecular weight degradation occurs at a faster rate when temperature is elevated to 160 C from 12 0 C. More importantly, with respect to the concentration of PLA loadi ng, time required to achieve complete degradation of PLA is reduced by almost se ven times at 120 minutes. This suggests that at 121 C there is likely a formation of intermediate produc ts that reduce the rate of hydrolysis. Tsuji et al., 2008 and Mohd-Adn an et al, 2008 suggested factors influence hydrolysis kinetics such as crystallinity and optical purity.

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29 Conclusions PLA hydrolysis is a complex reac tion. Temperature, concentration, molecular weight, crystallinity, size, thickness may af fect the reaction. A first order model was applied for this reaction. Values for activati on energy compared well to literature values.

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30 Figure 3-1. Cup used for obtaining feedstock

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31 0 20000 40000 60000 80000 100000 120000 140000 0100200300400500600700800 Time (minutes)MW (grams) 121 C 2.5 grams 121 C 7.5 gramsFigure 3-2. MW degradation 121C

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32 0 20000 40000 60000 80000 100000 120000 140000 020406080100120140 Time (minutes)MW (grams) 160 C 2.5 grams 160 C 7.5 gramsFigure 3-3. MW degradation 160C

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33 0 200 400 600 800 1000 1200 0100200300400500600700800 Time (minutes)Lactic Acid ( mM ) 121 C 2.5 grams 121 C 7.5 gramsA 0 200 400 600 800 1000 1200 0100200300400500600700800 Time (minutes)Lactic Acid (mM) 160 C 2.5 grams 160 C 7.5 gramsB Figure 3-4. Variation in lactic acid concent ration as a function of time. A) Lactic acid concentration while working at 121 oC. B) Lactic acid concentration while working at 160 oC

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34 modely = -0.0077x 0.677 R2 = 0.9722 -7 -6 -5 -4 -3 -2 -1 0 -1000100200300400500600700800 time (minutes)LN ( MWi-MW ) / ( MWi-MWf ) Experimental data-121C A modely = -0.059x 0.3767 R2 = 0.9723 -8 -7 -6 -5 -4 -3 -2 -1 0 020406080100120140 Time (minutes)LN (MW-MWf)/(MWf-MWi) Experimental data-160C B Figure 3-5. Linearized plot of rate of change with respect to time A) Model and Experimental data at 121 C B) Model and Experimental data at 160C

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35 y = -8907.7x + 17.742 R2 = 1 -6 -5 -4 -3 -2 -1 0 0.002250.00230.002350.00240.002450.00250.00255 Time(minutes)LN (k) Ea/R Linear (Ea/R) Figure 3-6. Determination of Ea for MW degradation

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36 modely = -0.0011x + 0.1663 R2 = 0.9328 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0100200300400500600700800 Time (minutes)LN ( LAf LA ) / ( LAf LAi ) 121 C 2.5 and 7.5 grams (average) A model y = -0.0129x + 0.3688 R2 = 0.9315 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0100200300400500600700800 Time (minutes)LN(LAf-LA)/(LAf-LAi ) 160 C 2.5 and 7.5 grams (average) B Figure 3-7. Lactic acid concentration plotted as first order equation A) 2.5 and 7.5 grams 121 C B) 2.5 and 7.5 grams 160C

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37 Table 3-1. Molecular weight degradation of PLA at 121C PLA (grams) 2.5 7.5 Time (min) Mw (g/mol) Mw (g/mol) 0 1.21 x 10 5 1.21 x 10 5 120 2.80 x 10 4 4.03 x 10 4 240 1.29 x 10 4 1.49 x 10 4 360 1.09 x 10 4 7.87 x 10 3 480 8.10 x 10 3 6.36 x 10 3 720 5.57 x 10 3 4.70 x 10 3

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38 Table 3-2. Molecular weig ht degradation of PLA 160C PLA (grams) 2.5 7.5 Time (min) Mw (g/mol) Mw (g/mol) 0 1.21 x 10 5 1.21 x 10 5 30 7.85 x 10 4 1.18 x 10 4 45 5.92 x 10 3 6.85 x 10 3 60 2.39 x 10 3 3.50 x 10 3 90 1.36 x 10 3 1.51 x 10 3 120 9.27 x 10 2 7.24 x 10 2

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39 Table 3-3. Molecular weight PL A degradation constant k and Ea k (min1 ) T (K) Ea (kJ) 7.7*103 394 74.1 5.9*102 433 Table 3-4. Lactic acid first order rate constants Mass (grams) Temp(C) k 2.5 7.5 (average) 121 1.10*103 2.5 7.5 (average) 160 1.29*102

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40 Table 3-5. Primary transition temper atures of selected PLA copolymersa Copolymer ratio Glass transition temp (C)Melting temperature ( C) 100/0 (L/D,L)-PLA 63 178 95/5 (L/D,L)-PLA 59 164 90/10 (L/D,L)-PLA 56 150 85/15 (L/D,L)-PLA 56 140 80/20 (L/D,L)-PLA 56 (125) b a Data from Garlotta, 2002 bMelting point achieved by strain crystallization

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41 Table 3-6. Effect of processing condition s on mechanical properties of PLA copolymersa Copolymer ratio Process condition Tensile strength Young's modulus Elongation (%) MW (L/D,L)PLA MPa GPa 100/0 Injection molded, crystallized 64.8 4.0 800000 90/10 Injection molded, amorphous 53.4 1.03 4.6 90/10 Injection molded, crystallized 58.6 1.29 5.1 90/10 Extruded, biaxially oriented, strain crystallized 80.9 3.41 41.2 145000 90/10 Extruded, biaxially oriented, strain crystallized, heat set 70.1 2.76 20.7 145000 95/5 Extruded, biaxially oriented, strain crystallized 68.6 1.88 56.7 120000 95/5 Extruded, biaxially oriented, strain crystallized, heat set 60.7 1.63 63.8 120000 80/20 Injection molded, amorphous 51.7 2.1 5.7 268000 80/20 Extruded, biaxially oriented, strain crystallized 84.1 2.9a4 18.2 268000 80/20 Extruded, biaxially oriented, strain crystallized, heat set 80.1 2.54 32.3 268000 a Data from Garlotta, 2002

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42 CHAPTER 4 ANAEROBIC DIGESTION OF HYDROLYZED AND NON-HYDROLYZED POLYLACTIC ACID Introduction Plastics play an important role as a resu lt of their many applications such as packaging. Howev er, because of thei r persistence in the environment, and the increased cost of solid waste disposal due to the reductions in available landfill space as well as the potential hazards from wa ste incineration, polymers have become a waste management problem. Thus, biodegradable polymers as a potential partial solution of these problems were developed during the last decade. Despite being a compostabl e polymer, currently PLA waste is being sent to landfills. Landfills do not offer an environment fo r efficient PLA degradation. Landfills are made to serve as a perennial containment fo r waste. Landfills are not just one compact mass of dirt; they are layers of dirt and wa stes. Landfills begin with a lining at the bottom (concrete or plastic), mineral se aling layer, a protective laye r, drain, drainage layer, and garbage being placed on the top of it, then a new layer of dirt and impermeable material is leveled for cover; thus minimizing mois ture ingress. Liquid collection systems are installed below and above the liners so that any leachate which leaks through or is retained on them can be recovered. Deposition of wet wastes is reduced to a practical minimum. In the first instance, landfill gas should be avoided as far as practicable as they represent a potential risk to people. Invariable material in the landfill begi ns to degrade over time generating landfill gas. Although landfill gas recovery is seen as one of the end-of-the-pipe solution to the problem of escaping landfill gas. The trend in a number of countries is to discourage or prohibit landfilling of organic wastes so t hat any future methane generation in the sites

PAGE 43

43 would be minimal or negligible. The cost of landfilling are high; a study done in the United Kingdom on urban and rural municipal waste disposal indicated that costs range between $ 11.25 and $ 33.75 per t on (UNEP, 1995) In conventional landfills due to the prolonged persistence of adverse conditions fo r microbial growth, it takes decades for the waste to degrade and yield methane. Mor eover, the gas production is not sustained and is subjected to temporal and spatial variations across the landfill. Anaerobic digestion technologies have been developed for accelerating the biological degradation of the wastes either in bioreacto r landfills or in-vessel systems. In anaerobic digestion process, organic compound like carbohydrates, fats and proteins are mineralized to biogas through the syntrophic action of several groups of microorganism. The process occurs in nature in anaerobic environm ents in the absence of molecular oxygen, like wetland s, rice fields, intestines of animals, aquatic sediments, and manures, and is responsible for car bon cycling in these environments. The engineered process is called anaerobic digestion (Lai et al., 2008). Anaerobic Digestion of PLA will help not only to save space and money, but it will produce some energy in exchange. Previous studies conducted by (Vargas et al., 2009) have proven that biodegradability of PLA can be in creased by hydrolysis of PLA. The Biochemical Methane Potential (BMP) assay is a procedure developed to determine the methane yield of an organic material during its anaerobic decomposition by a mixed microbial flora in a defined medium. This assay provides a simple means to monitor relative biodegradability of s ubstrates (Owens and Chynoweth, 1993).

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44 The aim of this study was to determine, the BMP of polylac tic acid that was hydrolyzed using different dur ation of heat exposure and co mparing the obtained results with available literatur e on anaerobic composting/digestion of PLA. Background The fermentation process in whic h or ganic material is degraded and biogas (composed of mainly methane and carbon dioxi de) is produced, is referred to as anaerobic digestion. Anaerobic digestion proc esses occur in many places where the organic material is available and redox potential is low (zero oxygen). The amount of excess sludge produced is very small and well stabilized, hence even the so called granular anaerobic sludge produced in the bioreactor has economic value (Van Lier et al, 2008). The complete mineralization to methane and CO2 of the polymer under anaerobic conditions involves successions of syntrophic associations (Schmotz et al., 2006). Hence, most anaerobic biodegra dation studies of polyesters have focused on mixed and unspecified populations such as divers e anaerobic sludges and sediments (Budwill et al., 1992; Reischwitz et al., 1998). From the literature, it was realized that anaerobi c biodegradation of PLA depends on temperature, microbial cu ltures, pH, agitation, surrou nding biomass, and whether the medium is liquid or solid (Abou-Zeid et al, 2001). Several reports showed that the crystalline part of the PLA was more resistant to degradation than the amorphous part, and that the rate of degradation decreases with an increase in crystallinity (Tsuji et al., 2001). The degradation behavior of polymers also depends on their number average mole cular mass (Mn) or weight average

PAGE 45

45 molecular mass (Mw). Polymer molecules, ev en of the same type, come in different sizes; so we have to take an average of so me kind (weight average molecular weight): Ni: number of molecules Mi: Molecular weight The number average molecular weight is the ordinary arithmet ic mean or average of the molecular weights of t he individual macromolecules. Ni: number of molecules Mi: Molecular weight High molecular weight polymers are degraded at a slower rate than those with low molecular weights (Tokiwa and Suzuki 1978). The melting temperature (Tm) of polyesters has a great effect on enzymatic degradability. The biodegradability of PLA depends on the environment to which it is exposed. In human or animals bodies, it is believed that PLA is initially degra ded by hydrolysis and the soluble oligomers form ed are metabolized by cells. Upon disposal in the environment, it is hydrolyzed into low molecu lar weight oligomers and then mineralized into CO2 and water by microorganisms present in the environment (Lunt 1998). Soil burial tests show that degradation of PLA in soil is slow and that it takes a long time for degradation to start. For instance, no degradat ion was observed on PLA sheets after 6 weeks in soil (Okhita and Lee 2006). On the other hand, PLA can be degraded in a composting environment where it is hydrol yzed into smaller molecules (oligomers,

PAGE 46

46 dimmers and monomers) after 45-60 days at 50-60 C. These smaller molecules are then degraded into CO2 and water by microorganisms in the compost (Tokiwa et al., 2006). PLA degraders have a limited di stribution and rather scarce in the soil environment compared with those that degr ade poly hydroxyl butyrate (PHB), poly caprolactone (PCL) and poly butylene succinate (PBS). The population of these polyester-degrading microbes decreased in the order of PHB = PCL > PBS > PLA. A burial test comparison of PLA and other polyesters, e.g. PHB, PCL and PBS, indicated that PLA was not readily degraded when the PLA samples were buried under soil for 20 months (Tokiwa and Jarerat, 2004). Microbial degradation of PLA was first published by Pranamuda et al. (1997) using an actinomycete Amycolatopsis strain isolated form soil. Si nce then, quite a number of Amycolatopsis strains have been isolated as PLA degraders. Ikura and Kudo (1999) analyzed 50 samples collected from soil, pond, rivers but only two strains were capable of degrading more than 50% of L-PLA film in the liquid medium. The sequence of the strain is closely related to Amicolatopsis mediterranei Another L-PLA degrading microorganism, Amycolaptosis sp strain K104-1 was isolated from 300 soil samples. In addition to Amycolaptosis several actinomycetes belonging to Lentzea, Kibdelosporangium Streptoalloteichus and Saccharothrix are also capable of degrading PLA. Out of 14 fungal strains te sted, only two strains of F. moniliforme and one strain of Penicillium roqueforti could assimilate lactic acid and ra cemic oligomer products of PLA, but no degradation was observed on PL A (Tokiwa and Calabia, 2006).

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47 The microbial vulnerability of polymers is at tributed to the biosynthesis of lipases, esterases, ureases and prot ases. Enzymes involved in deterioration require the presence of cofactors such as, presence of ca tions present in the material matrix and coenzymes synthesized by microorganisms, for the breakdown of specific bonds. The biodeterioration of thermoplastic polymers could proceed by two different mechanisms (i.e., bulk and surface erosion). PL A proceeds by bulk erosion, in the case of bulk erosion, fragments ar e lost from the entire polymer mass and the molecular weight changes due to the bond cleavage. This l ysis is provoked by chemicals such as, water, acid, bases, transition metals and radi cals, or by radiation but not by enzymes. They are too large to penetrate throughout the ma trix framework (Lucas et al., 2008). Several studies have shown that cert ain proteases, including proteinase K, pronase and bromelain have bee n found to increase the rate of degradation of PLA while estereases do not (Hakkarainen et al., 2002; MacDonald et al. 1996). Torres et al., 1999, found growth of fungal mycelia on racemic PLA plates after 8 weeks in soil. Urayama et al., 2002, found onl y a 20% decrease in molecular weight of PLA (100% L) plates after 20 months in soil while a 75% decrease was noted for PLA (70% L). Ho and Pometto 1997, found about 20% of a PLA film was mineralized to CO2 after 182 days in a laboratory respirometer charged with soil at 28C. It was found that PLA films had weight loses varying form 0 to 100% after burial in soil for 2 years depending on PLA type and locati on (Shogren et al, 2002). Vargas et al., (2009), found that PL A samples appeared to be much more vulnerable to themophilic anaerobic biological degradation when PLA was pretreated (gamma radiation, electron beam etc). However, at 37 C, untreated (no pretreatment)

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48 PLA showed negligible weight loss under anaerobic conditions after 180 days. Temperature of incubation was a key factor for anaerobic biodegradation of PLA. In this work methanogenesis was initiated after 21 da ys of incubation at 58C, and this was found to be the quickest breakdown of PLA based on literature review. This study focuses on direct hydrolysis of PLA at 160 C and mass loading of 2.5 grams of PLA and anaerobic degradation of hydrolyzate after pretreatment. Material and Methods Feedstock Waste was created using commercial t hermoformed cups (Fabri-Kal, Inc., Kalamazoo, MI) obtained from TR EEO Center at the University of Florida (Figure 3-1). Anaerobic Digestion Protocol The mixed microbial flora preparation and the PLA pretr eatment was carried out according to the following protocols. Mixed microbial flora (inocolum) preparation Vargas et al., 2009, reported good resu lts when thermally treated PLA and untreated PLA was s ubjected to anaerobic digestion using thermophilic conditions. In this experiment we first adapted the mixture of microbial flora (inoculum) to acidic feed (i.e. lactic acid) at thermophilic conditions. To adapt the microbial flora, a 5 L reacto r (digestor) was used (Figure 4-1). Using a batch operation the digestor wa s fed with silage sorghum (f ermented). It is well known that fermented sorghum develop organic acids such as lactic acid. After the digestion ceased lactic acid 80 % (L) was added to the digestor. Lactic acid was added daily and the concentration was slowly increased.

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49 The pH and gas produced by the digestor was measured daily. Aliquots of 2.5 mL of lactic acid addition per day was det ermined as maximum for the system. The inoculum was under adaptation for 210 days. Feedstock preparation The created waste was subjected to Hydr olysis. Three set of samples were prepared using duplic ates (Table 4-1). After the hydrolysis Molecular Weight and Lactic Acid content was measured in the samples. The samples with higher ratio of lactic acid recuperation were selected. Best results were achieved when working with concentration of 2.5 grams of PLA with 30 grams of water with thermal exposure of 160C (Table 4-2). BMP preparation The three hydrolyzed samples and untr eated PLA were placed in glass serum bottles (cap 280 mL). 100 mL of the adapted thermophilic inoculum and nutrients were added (Table 4-3). The bottles were seal ed with butyl rubber stopper and crimp with aluminum caps; set at 55C in a Lab-Line L-C Incubator (Lab-Line Instruments,Inc., Melrose Park,IL). Analysis Every 4 days, gas production was measur ed using a syringe and composition was determined using a Gas Partitioner Chro matograph model 1200 (Fisher Scientific Philadelphia, PA) adapted with a thermal conductivity detector. Biochemical methane potential (BMP) of PLA was expressed as a yield of methane per gram of PLA sample loaded into BMP bottles, and was determined in accordance to ASTM E1 196 (ASTM,1996). Also, Chemical Oxygen Demand (COD) was measured weekly as a way to know if the reaction was taking place or had stopped.

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50 Results Hydrolyzed PLA showed a faster digestion than raw P LA. Figure 4-2A compares the milliliters of methane produced per gram of hydrolyzed PLA. It can be seen that the slope of the samples differs depending on the time they were exposed to heat (Figure 43); also, they differ in the amount of methane produced. From all the hydrolyzed samples under anaerobic digestion; the samp le with 60 minutes of heat exposure showed better results. PLA samples with 120 minutes of heat exposure at 160 C showed a 16.34% of methane concentration after 4 days, reaching maximum methane concentration of 54.83% after 23 days. The total amount of methane produced by this sample was 44.92 mL CH4 /g PLA. PLA samples with 60 minutes of heat exposure at 160 C showed a 15.20% of methane after 4 days, reaching a maximum methane concentration of 58.47% after 38 days. The total amount of methane produced by this sample was 160.65 mL of CH4 / g PLA. PLA samples with 30 minutes of heat exposure at 160 C showed an 11.40% of methane concent ration after 4 days, reaching a maximum methane concentration of 49.03% after 27 days. Raw PLA digestion is shown in Figure 4-2B. Raw PLA samples without any heat treatment showed a 7.90% of methane concentration after 8 days, reaching a maximum methane concentration of 50.80% after 42 days. Discussion Previous experiments conducted by Var gas et al., 2009, reported for untreated (raw) PLA hydrolysis and acidific ation to occur during the first 21 days of anaerobic digestion, and a production of 187 cc CH4/g PLA after 56 days at 58 C. In this experiment we started to have methane production as soon as 4 days after the

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51 anaerobic digestion began and a production of 232 mL CH4 / g PLA after 54 days at 50 C. Theoretical methane yield was calculat ed to be 339 mL CH4/g PLA at standard conditions of temperature and pressure. This value was calculated using stoichiometry of the assumed reactions that governed the anaerobic process. C3H6O3 + 3 O2 3 O2 + 3 H2O 3 x 32 COD = 1.067 90 PLA 1.067 g COD x 0.35 L CH 4 = 373 x (298/328) = 339 mL CH4 / g PLA (STP) g PLA g COD For all the hydrolyzed PLA methane produc tion was calculated assuming 100% of lactic acid concentration in PL A. Therefore, in two grams a liquot of a 2.5 grams of PLA and 30 grams of water mixture the ex pected amount of methane would be: 2.5 g PLA x 2 g mixture = 0.154 g PLA 32.5 g mixture 0.154 g PLA x 1.067 g COD = 0.164 g COD g PLA 0.164 g COD x 0.35 L CH4 = 57.45 mL CH4 g COD Figure 4.2A shows experimental data colle cted when digesting hydrolyzed PLA for 30, 60 and 120 minutes.

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52 The performance of the BMPs was evaluat ed by fitting the cumulative methane production data to the modified Gompertz equation (Koppar and Pullammanappallil, 2008). The Gompertz equation describes cu mulative methane production from batch digesters assuming that methane production is a function of bacterial growth (Table 4.5) Samples with heat exposure of 120 and 60 minutes were completely done when these results were written. However, t he collected data in the other two BMPs, 30 minutes and raw, are good enough to predict the final me thane production; the final methane production were calculated to be 143.1 and 323.62 mL respectively. The methane production differs considerab ly between the samples treated 30 and 60 minutes to the one treated 120 minutes Also, the methane production differs considerably between the raw PLA and the samples treated 30 and 60 minutes. The difference in methane production between t he samples treated for 30 and 60 minutes are rather low. This difference in methane production may be due to the feedstock used in each of the BMPs. When digesting raw PLA a methane production of 339 mL of methane per gram of PLA was predicted; however, the act ual yield is 323.62 mL. The error between Gompertz model value and the t heoretical value is 4.5%. A t heoretical value of methane for samples with heat exposure of 30 and 60 mi nutes is hard to determine since in both cases the feedstock was part solid and part liquid. The PLA sample with heat exposure of 120 minutes was liquid; upon normalizing t he results the lactic acid production is 291.68 mL of methane per gram of PLA. Assu ming that the sample was just monomer (lactic acid) the expected gas production was 339 mL. The error between the

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53 experimental value and the theoretical value was 6.0%. In all the cases the methane production is less than the stoichiometic theor etical value. The theoretical value used was calculated assuming 100% of lactic acid concentration in the PLA used. Nevertheless, this is not the case. The amount of lactic acid in samples varies from 60 to 80% depending on fillers and plasticizers (Garlotta, 2001). The used fillers and plasticizers may have a different behavior than lactic acid in anaerobic digestion. These results suggested that adapted bacteria and thermophilic anaerobic digestion should be used when anaerobically di gesting PLA. From t he results hydrolysis pretreatment seems to expedite the r eaction, and more importantly, it can be understood that previous hydrolysis is not needed when using an adapted microbial flora. From literature we know that some stra ins of Actinobacteria (i.e. Amycolatopsis) and Firmicutes (i.e. Bacillus stearothermophilus, G eobacillus thermocatenulatus and Paenibacillus amylolyticus strain TB-13 ) have been effective when digesting PLA anaerobically. In a study of the UF therm ophilic inocolum used for this experiment Actinobacteria and Firmicutes were present in the mixed flora; however, the identification analysis does not i ndicate the presence of the fa mily of bacteria described in the literature. This suggested that the c oncentration of the particular bacteria was negligible when the adaptation of the bacte ria started, but separate adaptation can increase the concentration of appropriate bacteria. On th e other hand, it may be possible that more than one kind of Actinobacteria and Firmicutes are able to produce Proteases, Lipases and Estereases that are needed to break down PLA.

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54 Future Work To evacuate doubts whether if another Actinobacteria and Fir micutes are able to produce Proteases, Lipases and Estreases an identification study in the adapted incolum is suggested.

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55 Figure 4-1. Mixed micr obial flora adaptation

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56 0 50 100 150 200 250 300 350 0102030405060708090 Time (days)mL CH4/ g PLA h y drol y zed 160 C 120 min 160 C 60 min 160 C 30 minA 0 50 100 150 200 250 300 350 0102030405060708090 Time (days)mL CH4/ g PLA Raw PLAB Figure 4-2. PLA anaerobic digestion. A) Hydrolyzed PLA. B) Raw PLA.

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57 Figure 4-3 Initial methane producti on rate from hydrolyzed PLA. Figure 4-4 Methane production from hydrolyzed PLA and raw PLA in the first 21 days of anaerobic digestion.

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58 Table 4-1. Hydrolyzed selected samples PLA (grams) H2O (grams) Temperature (C) Retention time (min) Number 2.494 31.566 160.00 30.00 M16 2.584 30.549 160.00 60.00 M18 2.534 30.512 160.00 120.00 M20 2.61 31.336 160.00 30.00 M11 2.539 30.665 160.00 60.00 M13 2.552 30.665 160.00 120.00 M15 Table 4-2. Lactic acid concentration and per cent recovery of hydrolyzed selected samples Number Lactic Acid (mM) Recovery (%) M16 0.03 1.28 M18 0.32 12.26 M20 0.71 27.87 M11 0.03 1.14 M13 0.35 13.59 M15 0.78 30.68

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59 Table 4-3. Content of BMP bottles Number PLAhydrolyzed (grams) Inocolum (mL) RAW 2.0 100 M16 2.4 100 M18 2.1 100 M20 2.4 100 RAW 2.0 100 M11 2.1 100 M13 2.1 100 M15 2.1 100 Table 4-4. PLA molecular weight after hydrolysis Number MW(grams) RAW 1.21 x 10 5 M16 2.87 x 10 3 M18 7.93 x 10 2 M20 6.60 x 10 2 RAW 1.21 x 10 5 M11 3.60 x 10 3 M13 1.53 x 10 3 M15 8.22 x 10 2

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60 Table 4-5 Summary of performance of hydrolyzed and non hydrolyzed PLA Feedstock condition Temperature anaerobic digestion C CH4 yieldexperimental mL CH4/g PLA Gompertz parameters (model)a Duration to produce 95% CH4 yield potential P b Rm b b mL CH4 Kg VS-1 mL CH4 Kg VS-1day-1 days Raw PLA 55 323.62 6.99 25.07 86.92 30 minutespretreatment 55 143.1 2.4 0.64 83.48 60 minutespretreatment 55 160.65 158.28 5.67 3.47 85.25 120 minutespretreatment 55 44.92 42.23 3.14 2.79 40.11

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66 BIOGRAPHICAL SKETCH Cesar M. Moreira was born in Guayaquil, Ecuador. At the age of twenty-nine he moved to Miami Florida. He received his Associate of Arts Degree from Miami Dade College in 2005 and was admitted to the D epar tment of Agricult ural and Biological Engineering at the University of Florida. In 2007 he obtained hi s B.S. in Agricultural and Biological Engineering In 2008, he was admitted to the Graduate School at University of Florida to pursue a MS degree in the Department of Agricult ural and Biological E ngineering under the supervision of Dr. Bruce A Welt. He graduated with a masters degree in December 2009.