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Effect of Pre-Treatments on the Kinetics of Subsequent Aerobic and Anaerobic Biodegradation of Polylactic Acid (PLA)

Permanent Link: http://ufdc.ufl.edu/UFE0022101/00001

Material Information

Title: Effect of Pre-Treatments on the Kinetics of Subsequent Aerobic and Anaerobic Biodegradation of Polylactic Acid (PLA)
Physical Description: 1 online resource (108 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: anaerobic, biodegradable, biopolymer, compost, degradation, kinetics, packaging, pla, polylactic, post
Agricultural and Biological Engineering -- Dissertations, Academic -- UF
Genre: Agricultural and Biological Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The purpose of this work was to evaluate treatments that increase polylactic acid (PLA) biodegradation during composting and anaerobic digestion. Treatments were chosen on the potential to reduce molecular weight and affect structural integrity of the polymer. Five potential treatments were investigate, and it was found that steam exposure was the most effective treatment with up to 94% molecular weight reduction with 120?C steam for 4 hours. To determine kinetics of PLA molecular weight reduction after exposure to steam, a first-order reaction model was proposed. It was found that the model, which is time and temperature dependent, fits experimental data well, with activation energy, Ea, of 52.3 KJ/mol. In addition, it was demonstrated that steam treatments hydrolyze polymer molecules, resulting in de-polymerization to lactic acid. At 120?C, weight loss of PLA was 84.7% after 24 hours, indicating significant conversion to lactic acid. To develop a simple method to evaluate PLA aerobic biodegradation in compost, the fundamentals of conversion were examined, and three approaches were designed and assessed. These methods are referred to as methods of 'flexible bags,' 'rigid containers with plastic lids,' and 'perforated jars.' It was found that the method of perforated jars was most reliable, simple and consistent to apply. To evaluate effects of treatment in subsequent anaerobic digestion, weight loss and biochemical methane potential (BMP) of treated samples were investigated under mesophilic and thermophilic anaerobic conditions. Untreated PLA did not degrade under mesophilic conditions. However, degradation did occur under thermophilic conditions, producing 187 ccCH4/g at 58 degrees C after 56 days. The best scenario evaluated, was steam-treated PLA at 120 degrees C for 3 hours subjected to anaerobic thermophilic conditions, where the yield of methane was 225 ccCH4/g after 56 days. While untreated PLA biodegrades more slowly than common organic feedstock, steam-treated PLA biodegraded faster. Results of this investigation show that treatment of PLA with steam at 120 degrees C for 3 hours reached 60% degradation in 14 days. Compost was not altered by PLA conversion.
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.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Welt, Bruce A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022101:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022101/00001

Material Information

Title: Effect of Pre-Treatments on the Kinetics of Subsequent Aerobic and Anaerobic Biodegradation of Polylactic Acid (PLA)
Physical Description: 1 online resource (108 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: anaerobic, biodegradable, biopolymer, compost, degradation, kinetics, packaging, pla, polylactic, post
Agricultural and Biological Engineering -- Dissertations, Academic -- UF
Genre: Agricultural and Biological Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The purpose of this work was to evaluate treatments that increase polylactic acid (PLA) biodegradation during composting and anaerobic digestion. Treatments were chosen on the potential to reduce molecular weight and affect structural integrity of the polymer. Five potential treatments were investigate, and it was found that steam exposure was the most effective treatment with up to 94% molecular weight reduction with 120?C steam for 4 hours. To determine kinetics of PLA molecular weight reduction after exposure to steam, a first-order reaction model was proposed. It was found that the model, which is time and temperature dependent, fits experimental data well, with activation energy, Ea, of 52.3 KJ/mol. In addition, it was demonstrated that steam treatments hydrolyze polymer molecules, resulting in de-polymerization to lactic acid. At 120?C, weight loss of PLA was 84.7% after 24 hours, indicating significant conversion to lactic acid. To develop a simple method to evaluate PLA aerobic biodegradation in compost, the fundamentals of conversion were examined, and three approaches were designed and assessed. These methods are referred to as methods of 'flexible bags,' 'rigid containers with plastic lids,' and 'perforated jars.' It was found that the method of perforated jars was most reliable, simple and consistent to apply. To evaluate effects of treatment in subsequent anaerobic digestion, weight loss and biochemical methane potential (BMP) of treated samples were investigated under mesophilic and thermophilic anaerobic conditions. Untreated PLA did not degrade under mesophilic conditions. However, degradation did occur under thermophilic conditions, producing 187 ccCH4/g at 58 degrees C after 56 days. The best scenario evaluated, was steam-treated PLA at 120 degrees C for 3 hours subjected to anaerobic thermophilic conditions, where the yield of methane was 225 ccCH4/g after 56 days. While untreated PLA biodegrades more slowly than common organic feedstock, steam-treated PLA biodegraded faster. Results of this investigation show that treatment of PLA with steam at 120 degrees C for 3 hours reached 60% degradation in 14 days. Compost was not altered by PLA conversion.
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.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Welt, Bruce A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022101:00001


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80909f36728202356116966c23bc965d
584023e7f0a3c04850ac4e63bcb0118bfcce08fd







EFFECT OF PRE-TREATMENTS ON THE KINETICS OF SUBSEQUENT AEROBIC AND
ANAEROBIC BIODEGRADATION OF POLYLACTIC ACID (PLA)



















By

LUIS FERNANDO VARGAS DELGADO


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2008


































2008 Luis Ferando Vargas Delgado



































To my wife, son and parents









ACKNOWLEDGMENTS

I express my sincere appreciation to my advisor Dr. Bruce Welt. Definitely, he is an

exceptional professor and a good friend, very dedicated and always happy to support me with his

valuable guidance. I am grateful to my committee members Dr. Art Teixeira, Dr. Balaban, Dr.

Pullammanappallil, and Dr. Beatty for their orientation. I thank my colleagues Richlet Dorcent,

Ayman Abdellatief and Cecilia Amador for their collaboration and friendship. I express my

gratitude to Steve Feagle, James Rummell and Billy Duckworth whose technical expertise was

very valuable for this research. Also, I was happy to interact with my colleagues from the

bioprocess engineering lab, who were always available to assist me. I show my sincere

appreciation to the University of Florida and the Agricultural and Biological Engineering

Department for their funding and support.









TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

L IST O F T A B L E S ...................... ............... ....................................................... . 8

LIST OF FIGURES .................................. .. ..... ..... ................. .9

L IST O F A B B R E V IA TIO N S ...................... .. .. .......... ............................................ 12

A B S T R A C T ............ ................... ............................................................ 14

CHAPTER

1 IN T R O D U C T IO N ...................................................... ................................................ .. 16

2 EVALUATION OF TREATMENTS TO REDUCE PLA MOLECULAR WEIGHT ............18

In tro du ctio n ................... .......................................................... ................ 18
M materials and M methods ...................................... .. .......... ....... ...... 19
M materials ...............................................................................................19
M ethods of A analysis ............................. ..... .. ................ ..... .. ...... ........ ......19
Subjective assessment of structural integrity ....................................................19
D term nation of w eight loss ............................ .. .............. ...........................19
Determination of molecular weight............ ..................... ............... 20
Determination of degree of chain scission .................................... ............... 20
Determination of mechanical properties ............................ ..... ..................21
Exposure of PLA to Gam m a Irradiation .................................. .................................... 21
Exposure of PLA to Electron Beam Irradiation ............................................................21
Immersion of PLA in Alkaline M edia................................ ........................ ......... 23
Im m version of PLA in A cid M edia........................................................ ............... 24
E exposure of P L A to Steam .................................................................. .... .................. 24
R results and D discussion ........................................................................ .... 24
Exposure of PLA to Gam m a Irradiation .................................... .................................. 24
M echanical properties ........................................... .................. ............... 24
M molecular w eight .................. ........................................................ .. 25
Degree of chain scission.......................................................... 26
Exposure of PLA to Electron Beam Irradiation ............................................................26
B before com posting .............................. .... ....................... ... ...... .... ..... ...... 26
Stru ctural integrity ......... ....... ............................................................. .... .... ... .. 2 6
M molecular w eight .................. ...................... ..... .................. .... 27
D egree of chain scission................................................. .............................. 27
A after co m p o stin g ............ .............................................................. .. .... .... .. 2 8
Stru ctu ral integ rity .......... .................................................................. ........ .. ....... .. 2 8
W eight loss ................................. .................................. .......... 29
M molecular w eight .................. ................................. ........ ............. 30









D -v a lu e ............................................................................................................... 3 0
Im m version of PLA in Alkaline M edia...................................... ...................... ........... 31
Structural integrity ......... ....... ...................................................................... .. .... 1
W e ig h t lo ss ..................................................................................................... 3 1
M molecular w eight .................. ..................................... ................. 32
Im m version of PLA in A cid M edia........................................................ ............... 32
Stru ctural integrity ......... ....... ............................................................. .... .... ... .. 32
W e ig h t lo ss ..................................................................................................... 3 2
E exposure of PL A to Steam .................................................................. .... .................. 32
Structural integrity ......... ....... ...................................................................... .. .. 32
M molecular weight ............................................ ...................... ......... 33
C onclu sions.......... ..........................................................34

3 KINETICS OF REDUCTION OF MOLECULAR WEIGHT IN STEAM-TREATED
P L A ....... ...................................... .................................................... 4 7

Introduction ...................................................................................... 47
M methods ....................... ....................................... ........ 47
First Order Reaction Model ........................ ......... ........ ....... ..............47
Depolymerization of PLA to Lactic Acid Resulting from Steam Exposure ...................48
R e su lts an d D iscu ssio n ..................................................................................................... 4 9
First-O order R action M odel ................ ............. ......................................................... 49
Depolymeryzation of PLA to Lactic Acid Resulting from Steam Exposure ..................49
Conclusion ..................................... ........ ... 50

4 DEVELOPMENT OF METHODS TO EVALUATE AEROBIC BIODEGRADATION
O F P L A ......... ................................................................. 54

In tro d u ctio n ................... ...................5...................4..........
M materials and M methods ...................................... .. .......... ....... ...... 55
M ethod of Flexible B ags ................................................... .... ............................... 55
Method of Rigid Containers with Plastic Film Lids......................................................57
M ethod of P erforated Jars .................................................................. .... ...................58
R results and D iscu ssions......... ........................................................................ ........ ..... ...59
M ethod of Flexible B ags ................................................... .... ............................... 59
Method of Rigid Containers with Plastic Film Lids ...................................................61
M ethod of P erforated Jars .................................................................. .... ...................6 1
C o n clu sio n ................... ...................6...................2..........

5 BIODEGRADATION OF TREATED PLA UNDER ANAEROBIC CONDITIONS ............71

In tro du ctio n ........................71.....................................
M materials and M ethods ............... ...................... .. ....................... .. .............. 72
Weight loss of PLA in Water under Anoxic Conditions...........................................72
Weight Loss of Irradiated PLA in Anaerobic Biological Media.................................72
Biochemical Methane Potential (BMP) of Steam-Treated PLA in Anaerobic
B biological M edia.................................................. 73



6









R results and D discussion ................. ... ......... ..... ............ ... ............. .. ..............74
Weight Loss of PLA in Water Under Anoxic Conditions.............................................74
Weight Loss of Irradiated PLA in Anaerobic Biological Media.................................74
Biochemical Methane Potential (BMP) of Steam-Treated PLA in Anaerobic
B biological M edia........................................................ 75
C onclu sions.......... ..........................................................76

6 BIODEGRADATION OF STEAM-TREATED PLA UNDER COMPOSTING
C O N D IT IO N S ............................................................................... 80

In tro d u ctio n ................... ...................8...................0..........
M material and M ethods ................... ..... ........... .............................. ........... .... 81
Steam-Treated PLA Biodegradation in Compost.............. ................................81
Kinetics of Steam-Treated PLA Biodegradation in Compost...................................82
Weight Loss of Steam-Treated PLA in Compost and Comparison with other
Com m on Feedstock .................................... ..... .......... .............. .. 82
R results and D discussion ................. ...... ....... ...... .................. ... ............. ..............83
Steam-Treated PLA Biodegradation in Compost.............. ................................83
Kinetics of Steam-Treated PLA Biodegradation in Compost...................................84
Weight Loss of Steam-Treated PLA in Compost and Comparison with other
Com m on Feedstock .................................... ..... .......... .............. .. 85
C onclu sion .......... .... ................................................. ...........................85

7 CON CLU SION S......... .......... .................................... .. ......................92

8 RECOMMENDATIONS FOR FUTURE WORK ....................................... ............... 95

APPENDIX

A CHARACTERISTICS OF THE COMPOST ........................................ ....................... 96

B PERMEABILITY TO CARBON DIOXIDE................................................................. 97

C V O L U M E O F B IO M A SS .............................................................................. .....................99

D B A G V O LU M E ........................................ ............................................ ........ .. 100

E OVERALL COEFFICIENT OF DIFFUSION......................... ...............101

F NUTRIENT FORMULA FOR ANAEROBIC MEDIA........................................... ...............103

L IST O F R E F E R E N C E S ......... ............... ............................................................................... 104

BIO GR A PH ICA L SK ETCH ......... ..... ........... ............. ................................ ................... .... 108









LIST OF TABLES


Table page

2-1 Linear regression outputs and D-values of e-beam irradiated/composted PLA ...............46

2-2 Comparison of treatments to reduce PLA molecular weight...........................................46

4-1 Biodegradation of PLA by method of flexible bags ................ .... .................69

4-2 Biodegradation of PLA by the method of perforated jars ......... ..................................69

5-1 Production of methane in steam-treated PLA................... ............... .. ..............79

5-2 Weight loss of untreated PLA in water under anoxic conditions ................................79

6-1 pH of biomass (compost + biodegraded PLA) ..... ......... ......................................... 91

6-2 Parameters of the logistic model (%biod = a / (1 + (t/xo)-b) ................... ...................91

A-1 Characteristics of prepared mature compost.................. ........ ................. ............... .96

E-l Carbon dioxide effusion rate estimation through 5-hole lid ................. ................102

F- A naerobic m edia form ulation ................................................ .............................. 103









LIST OF FIGURES


Figure page

2-1 Drinking cups m ade of PLA ....................................................................... 35

2-2 Principle of intrinsic viscosity method for determination of molecular weight ...............35

2-3 Stress at break of y-irradiated PLA samples ..................................... ........ ............... 36

2-4 Strain at break of y-irradiated PLA samples ..................................... ........ ............... 36

2-5 Molecular weight of y-irradiated PLA samples ........................................... ...........37

2-6 Determination of G, in PLA y-irradiation..................... ..... ......................... 37

2-7 E-beam irradiated PLA showing voids formation .................................. ...............38

2-8 M molecular weight of e-beam irradiated PLA ........................................ ............... 38

2-9 Determination of G, in PLA e-beam irradiation...........................................................39

2-10 Tem perature profile of com post bulk ........................................... ......................... 39

2-11 E-beam irradiated PLA after 6 weeks in com post ........................................ ..................40

2-12 Structural integrity of e-beam irradiated/composted PLA............... .........................40

2-13 Weight loss of e-beam irradiated/composted PLA ........................................................41

2-14 Molecular weight of e-beam irradiated/composted PLA............................................41

2-15 H y droly sis of P L A ..................................................................... ........... ....................42

2-16 Plots of e-beam irradiation dose vs. log f,, for D-values estimation ......................... 42

2-17 Structural integrity of PLA subjected to alkaline media ............... ............. ...............43

2-18 Weight loss of PLA subjected to 0.1NNaOH........ ........ .... ........................ 43

2-19 Molecular weight of PLA subjected to alkaline media.......................... ............... 44

2-20 Structural integrity of steam-treated PLA ..................................... ........................ 44

2-2 1 P ores in steam -treated PL A ....................................................................... ..................45

2-22 M olecular weight of steam-treated PLA ................................ ......................... ....... 45

3-1 First-order reaction plots for steam-treated PLA..........................................................51









3-2 Arrhenius plot for steam -treated PLA ....................................................... .............. 51

3-3 Spectra of lactic acid obtained by FTIR-ATR......... ............................ ............... 52

3-4 Mass balance of PLA treated with steam at extreme conditions .................. ............52

4-1 Set up for plastic biodegradation assessment in compost ASTM D5538........................ 63

4-2 Interactions in flexible bags ......... ......... ........... ............................................63

4-3 Pictures of flexible bags with biomass biodegrading ..................................................64

4-4 C hem ical form ula of PL A ................................................................................ ...... ...64

4-5 Interactions in rigid container with plastic film lid................................. ............... 65

4-6 Picture of rigid container with plastic film lid............................................................65

4-7 Interaction in perforated jar .................................................................... .... .................66

4-8 Picture of jar filled with biomass and perforated lid............... ........ .................. 66

4-9 Picture of compost plus PLA after 40 days, in and out of the bag ................................67

4-10 Biodegradation of PLA using the method of flexible bags............... ............................67

4-11 Biodegradation of PLA using the method of rigid containers with plastic film lids.........68

4-12 Overall effective coefficient of diffusion for CO2 through 5 holes .................................68

4-13 Biodegradation of PLA using the method of perforated jars...........................................69

5-1 Supposed anaerobic reactions for PLA anaerobic biodegradation ....................................77

5-2 BM P bottle w ith anaerobic m edia........................................................... ............... 77

5-3 Steam -treated ground PL A ........................................................................ ..................77

5-4 W eight loss of irradiated PL A ......................................... ............................. ...............78

5-5 Conversion of steam-treated PLA (1200C x 3h) to CH4 under anaerobic conditions .......78

6-1 Main reactions in PLA biodegradation................. ............. ....................86

6-2 "H ead start" effect .......... ........ .................................. ........ ........ ........ 87

6-3 "Acceleration" effect ................................. .. ..... .. ........... ......... 87

6-4 Biodegradation of steam-treated PLA over time in compost........................ ...............88









6-5 Biodegraded PLA in compost ....................................................................... 88

6-6 Logistic model fit for PLA biodegradation data.......................................................... 89

6-7 Weight loss of steam-treated PLA in compost compared with corrugated board and
w o o d ........... .... ............... ...................................... .... .................................89

6-8 Corrugated paperboard subjected to compost for 14 days....................... ..............90

6-9 Wood subjected to compost for 14 days ................................................ 90

6-10 Steam treated PLA (1200C x 3h) subjected to compost for 14 days ..............................90

B-l Mounted system to determine CO2 transmission rate.......................................................98

B-2 Arrhenius plot for activation energy determination (CO2 permeability)...........................98

D-1 Volume-meter designed for bag volume determination. ............................................100

E-1 Carbon dioxide concentration in headspace over time ...................................................102









LIST OF ABBREVIATIONS


A Area (cm2)

D Irradiation dose (kGy)

Def Coefficient of diffusion (mol C02/h/%)

E Thickness of film (mil)

Ea Activation energy (KJ/mol)

Gs Degree of chain scission (w/o units)

k Reaction rate constant or kinetic constant (h-1)

ko Pre-exponential factor (h-1)

M : Molecular weight (g/mol)

Mn Number-average molecular weight (g/mol)

Mn o Number-average molecular weight, initial (g/mol)

My Viscosity-average molecular weight (g/mol)

Mw Weight-average molecular weight (g/mol)

NA Avogadro number = 1.023 x 1023

P Permeability (mol-mil/atm-day-m2)

Pco2 Permeability to CO2 (mol-mil/atm-day-m2)

Po Pre-exponential factor for permeability (mol-mil/atm-day-m2)

Patm Pressure, atmospheric (atm)

pco2 Partial pressure of CO2 (atm)

pvap Vapor pressure (atm)

R Gas Law constant (82.057 atm-cc/molK or 8.31 J/molK)

T Temperature, absolute (K)

t Time (h or days)









V Volume (cc)

Vhs Volume of headspace (cc)

Vbag Volume of bag (cc)

Vbag tot Volume of bag, total (cc)

Vfirm Volume of film (cc)

W Weight (g)

Wo Weight, initial (g)

Wf Weight, final (g)

w Weight loss (%)

p Density (g/cc)

r : Number of moles

hiredd Reduced viscosity (ml/g)

[/]: Intrinsic viscosity (ml/g)

% CO2 Percentage of CO2 (%)

[CO2 Concentration of CO2 (cc CO2/ cc total)









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

EFFECT OF PRE-TREATMENTS ON THE KINETICS OF SUBSEQUENT AEROBIC AND
ANAEROBIC BIODEGRADATION OF POLYLACTIC ACID (PLA)


By

Luis Fernando Vargas Delgado

May, 2008

Chair: Bruce Welt
Major: Agricultural and Biological Engineering


The purpose of this work was to evaluate treatments that increase polylactic acid (PLA)

biodegradation during composting and anaerobic digestion. Treatments were chosen on the

potential to reduce molecular weight and affect structural integrity of the polymer. Five potential

treatments were investigate, and it was found that steam exposure was the most effective

treatment with up to 94% molecular weight reduction with 1200C steam for 4 hours.

To determine kinetics of PLA molecular weight reduction after exposure to steam, a first-

order reaction model was proposed. It was found that the model, which is time and temperature

dependent, fits experimental data well, with activation energy, Ea, of 52.3 KJ/mol. In addition, it

was demonstrated that steam treatments hydrolyze polymer molecules, resulting in de-

polymerization to lactic acid. At 1200C, weight loss of PLA was 84.7% after 24 hours, indicating

significant conversion to lactic acid.

To develop a simple method to evaluate PLA aerobic biodegradation in compost, the

fundamentals of conversion were examined, and three approaches were designed and assessed.

These methods are referred to as methods of "flexible bags", "rigid containers with plastic lids"









and "perforated jars". It was found that the method of perforated jars was most reliable, simple

and consistent to apply.

To evaluate effects of treatment in subsequent anaerobic digestion, weight loss and

biochemical methane potential (BMP) of treated samples were investigated under mesophilic and

thermophilic anaerobic conditions. Untreated PLA did not degrade under mesophilic conditions.

However, degradation did occur under thermophilic conditions, producing 187 ccCH4/g at 58C

after 56 days. The best scenario evaluated, was steam-treated PLA at 1200C for 3 hours

subjected to anaerobic thermophilic conditions, where the yield of methane was 225 ccCH4/g

after 56 days.

While untreated PLA biodegrades more slowly than common organic feedstock, steam-

treated PLA biodegraded faster. Results of this investigation show that treatment of PLA with

steam at 1200C for 3 hours reached 60% degradation in 14 days. Compost was not altered by

PLA conversion.









CHAPTER 1
INTRODUCTION

Recent efforts to improve sustainability imply, among other things, using renewable

resources, reducing municipal waste and avoiding solid, liquid (including oils) and gaseous

emissions [1]. Plastic packaging, while providing valuable contributions to society is often

perceived as a solid waste burden. Traditional plastic packaging does contribute to virtually non-

degradable solid waste in landfills. In addition, most polymers are petroleum-based, and

therefore, obtained from non-renewable resources. These issues have sparked interest in

biobased and biodegradable plastics [2].

Polylactic acid (PLA) is a biodegradable plastic polymer derived from renewable

resources, such as corn, that can be used in a variety of packaging applications. During

industrial composting, PLA is assumed to degrade through biological activity to carbon dioxide

and water. Compostability of PLA has been discussed by different authors, and the agreement is

that it truly occurs at temperatures around 580C after several weeks [3, 4, 5]. These

characteristics coupled with recent capital investments to produce commercial quantities of the

material have made PLA a viable option for replacing fossil fuel derived polymers for certain

applications [6].

As increasing amounts of PLA are finding packaging applications, a number of practical

challenges have become apparent including PLA's instability at moderate temperature and

relative humidity [7, 8], and PLA's relatively low biodegradation rate relative to typically

composted organic wastes, which is preventing commercial compost operators from accepting

PLA. Therefore, most post-consumer PLA is being sent to landfills for disposal where

breakdown does not readily occur [9]. Since companies promote PLA's sustainable qualities,

consumers expect that PLA waste will be either composted or recycled, thus completing the









sustainable cycle. Sending PLA to sanitary landfills threatens to violate consumer and public

trust in sustainable packaging materials.

Biodegradation is a two-step process. First, polymer molecules are hydrolyzed into smaller

pieces resulting in lower molecular weight polymers, oligomers and monomers. Second,

biological action of microorganisms metabolizes these smaller molecules resulting in conversion

to carbon dioxide and water [10]. Therefore, it is suspected that treatments capable of rapidly

reducing PLA molecular weight will allow biological conversion sooner, which should lead to an

overall reduction in biodegradation time.

The motivating hypothesis of this work is that treatments capable of disrupting the polymer

matrix and/or reducing molecular weight should result in reduced overall composting time.

Additionally, in the event that composting time is reduced, experiments were designed to

determine whether pretreatments accelerated conversion kinetics or simply provided a head start

to the composting process that subsequently proceeded at the typical rate. Treated PLA will

already be constituted by smaller molecules when added into the composer, so less time is

needed to get to this point. The acceleration concept is based on increased metabolic activity

associated at higher concentrations of lower molecular weight polymer.

The main objective of this research was to evaluate effects of potential pretreatments on

kinetics of subsequent PLA aerobic and anaerobic biodegradation, and to determine whether or

not treatments will allow PLA to completely degrade within the time frame of normal organic

feedstock. Secondary objectives were to develop a methodology to measure PLA biodegradation

rate in a composting process and to determine kinetics of molecular weight reduction in treated

PLA.









CHAPTER 2
EVALUATION OF TREATMENTS TO REDUCE PLA MOLECULAR WEIGHT

Introduction

Composting is a well known process that is mainly used to break down organic materials

such as yard and food wastes. While PLA is susceptible to breakdown during composting, it has

been found that PLA degradation kinetics are considerably slower than typical organic

feedstocks, and currently, little, if any PLA waste is sent to industrial composting facilities.

Commercial PLA packages have been shown to be incompletely degraded after 28 days of

composting [3, 4] and recycling is currently not taking place [5]. Therefore, PLA represents a

potential bottleneck to composting operations, which could result in PLA being diverted to

sanitary landfills for disposal. In this regard, PLA's promise of sustainability would not be

completely fulfilled. Therefore, a key question for compostable sustainable plastics is how to

improve degradation kinetics without compromising important useful qualities of the polymer.

Some authors have reported on effects of gamma irradiation [11 ], electron beam irradiation

[12], enzymatic hydrolysis [13] and chemical hydrolysis on poly (L-lactide) acid (PLLA) [14].

Results showed reductions in molecular weight and associated loss of tensile strength, which are

indicators of polymer degradation. It is believed that such initial damage to the polymer will help

to at least provide a head start and may actually accelerate degradation kinetics of PLA during

composting.

This chapter evaluates potential treatments capable of reducing PLA molecular weight. For

this purpose, five treatments were chosen: (1) exposure to gamma irradiation, (2) exposure to

electron beam irradiation, (3) immersion in alkaline media, (4) immersion in acid media and (5)

exposure to saturated steam. Additionally, other analyses such as structural integrity and weight









loss were also performed. These treatments were compared and the most effective was selected

for further investigation under composting conditions (Chapter 6).

Materials and Methods

Materials

Thermoformed PLA drinking cups (Fabri-Kal, Inc., Kalamazoo, MI) were obtained from

TREEO Center at the University of Florida (Figure 2-1). Cup dimensions were measured using a

caliper (Mitutoyo Model CD-6 CS, Mitutoyo Corp., Japan) and are provided in Figure 2-1. Wall

thicknesses were 150-200m and bottoms were about 750[m.

Methods of Analysis

Subjective assessment of structural integrity

Polymer appearance and physical characteristics changed significantly due to various

treatments. Descriptive observation was used to compare samples at specific times during each

study. Assessment consisted of evaluation by touch, sight, physical attributes such as brittleness,

opacity, whitening, voids/pores formation, swelling, twisting, curling, conversion to powder and

weakening. A digital microscope (INTEL model APB-24221-99A, Mattel Inc., China) at

magnifications of 10X, 60X and 200X was used to enhance observational power.

Determination of weight loss

Weight loss (w) was calculated as a percentage using Equation 2-1.

W -W
w = x100% (2-1)


A precision scale (Voyager Pro/Ohaus, Ohaus Corp., Pine Brook, NJ) was used for

individual weight measurements. In all cases at least three repetitions were performed, but since

there was too much variability, only averages are presented in the graph. PLA samples were

carefully selected, cleaned and dried.









Determination of molecular weight

Intrinsic viscosity was used to determine weight-average molecular weight, 1 f,, and

viscosity-average molecular weight, My, of PLA samples in accordance to ASTM D2857-95

[15]. Kinematic viscosities, u, of PLA dilutions (up to 1% V/V)) were determined at 300C using

chloroform as solvent and a calibrated capillary viscosimeter (Cannon-Ubbelodhe Type N25,

State College, PA). These values were used to determine reduced viscosity, yred, and intrinsic

viscosity, [p], of each treated sample. Finally, molecular weight, M, was estimated using the

Mark-Houwink model (Equation 2-2), which relates molecular weight to the intrinsic viscosity.

Figure 2-2 shows the principle of this method.

[/i]= kM0 (2-2)

Constants, k and a, used for PLA in chloroform at 300C were 0.013 Iml/g and 0.777 for M,

and, 0.0153ml/g and 0.759 forMw [16]. At least two repetitions were performed for analysis of

gamma and e-beam irradiated (not composted) PLA.

Determination of degree of chain scission

It is well known that irradiation of polymers causes two main and opposing effects

including, scission and crosslinking. The important physical fact is that one chain scission causes

one molecule to become two, and one crosslink causes two molecules to become one [17]. To

evaluate the predominance of chain scission rather than crosslinking, the concept of degree of

chain scission, Gs, was applied. This is defined as the number of radiolysis events caused by the

absorption of 100eV of radiation and can be calculated by Equation 2-3 [11]. Number-average

molecular weight, Mn, was assumed to be equal to viscosity-average molecular weight, M#, since

doses were below the gel dose [18]. High values of G, indicate chain scission predominance over

crosslinkings.









1 I
NA(M MM
Gs =
6.24x 1016D (2-3)


Determination of mechanical properties

A 4301-Series Instron was used to determine stress and strain at break at a temperature of

250C in PLA samples. The equipment was set at a crosshead speed of 30 mm/min, and samples

set in the machine direction with a gage length of 30 mm. At least five repetitions were

performed in each tensile test to obtain representative average values. This analysis was

conducted for PLA samples exposed to gamma irradiation.

Exposure of PLA to Gamma Irradiation

Rectangular thin sheets (50mm x 10mm x 150-200[tm) and shredded pieces (~4mm edge)

obtained from PLA cup walls (Figure 2-1) were prepared and irradiated in a foil lined

paperboard canister (73 mm diameter x 180 mm height). The canister was placed inside the

irradiation chamber (FL Accelerator Services and Technology, Gainesville, FL) where it was

exposed to y-rays from a Cesium-137 source at a dose rate of 0.78 kGy/h. Samples were

irradiated in air in order to promote scission over crosslinking [11, 17]. The canister was

removed from the irradiation chamber after 92 and 221 hours. Dosimetry showed that samples

achieved absorbed doses of 72 kGy and 172 kGy, respectively. Irradiated samples were kept in

storage at 250C for at least 4 days prior to analysis. Mechanical properties (stress and strain at

break), molecular weight and degree of chain scission were subsequently assessed.

Exposure of PLA to Electron Beam Irradiation

Electron beam irradiation was applied to PLA samples in order to evaluate effects before

and after composting. Square sheets (31.5mm x 31.5 mm x 0.75mm) obtained from flat bottoms

of PLA drinking cups (Figure 2-1) were prepared and subjected to electron beam irradiation (FL









Accelerator Services and Technology, Gainesville, FL) at a dose rate of 2.4 kGy/min. Bunches of

PLA samples were placed together in Petri dishes that resulted in less than 1 cm overall thickness

[19]. Samples were transported by conveyor into the irradiation chamber for each irradiation

pass. Multiple passes were required to achieve desired doses. Since the goal was to induce chain

scissions, and since scission is aided by oxygen [11], no effort was made to protect samples from

oxygen. Samples were removed from the irradiation tunnel after 2, 4 and 6 passes at 36kGy per

pass, achieving absorbed doses of 72, 144 and 216 kGy. Irradiated samples were kept in storage

at 250C at least 4 days prior to analysis. Electron beam irradiated samples were evaluated for

structural integrity, molecular weight and degree of chain scission.

After e-beam irradiation, PLA samples were subjected to composting. For this purpose,

individual, previously e-beam irradiated samples, were placed inside hand-made, heat sealed

nylon screen envelopes and placed in a rotational composer (CompostTwin, Mantis,

Southampton, PA) equipped with thermocouples in a computer controlled environmental

chamber (Environmental Growth Chambers, Chagrin Falls, OH) at 350C. A standard organic

feedstock recipe was developed for this study and used throughout. Organic feedstock consisted

of freshly cut grass from the University of Florida's golf course (58%, C:N=10), saw dust (11%,

C:N=500), virgin corrugated paperboard (11%, C:N=170) and mature compost (20%). Raw

materials were sliced or cut in order to achieve a homogeneous mixture. Also, this formulation

met the requirement of initial optimum nutrient balance carbon/nitrogen ratio of 30. Water was

periodically added to the mixture in order to maintain moisture content between 55% and 70%,

and to promote and maintain biological activity [20]. Initial net weight in the composer was

about 118 lb and was rotated daily to ensure adequate mixing and aeration.









Screened sample envelopes allowed for intimate contact between samples and bulk

compost and protected against loss of non-mineralized sample during critical phases of

decomposition. Thermocouples inside the composer measured temperature of the composting

biomass over 10 weeks. Structural integrity, weight loss, molecular weight and irradiation

induced molecular weight loss D-value were determined in PLA samples for assessment of

electron beam irradiation effect on them.

Irradiation D-values are often used to describe microbial inactivation. A D-value

represents the dose required to cause one log cycle change in a measured parameter [21, 22, 23],

which means reducing its value to its 10%. Here, we used D-value to describe the irradiation

dose required to change molecular weight by one log cycle. D-values were calculated for

composted samples after 0, 1, 2 and 6 weeks. D-values were determined from inverses of slopes

from linear regressions of logo Mw versus absorbed dose. Therefore, steeper curves represent

lower D-values and greater sensitivity of molecular weight to dose.

Immersion of PLA in Alkaline Media

Rectangular sheets (6cm x 4cm x 0.75mm) obtained from PLA drinking cup bottoms

(Figure 2-1) were placed in 50 ml 0.1N NaOH and 50 ml of IN NaOH, and stored for 22 days at

250C. Samples immersed in 0. IN NaOH were evaluated over time for structural integrity, weight

loss, and weight-average molecular weight. Initially, the experimental design considered

evaluating both conditions (0.1N and IN), however, PLA samples immersed in IN NaOH

fragmented in such small parts that it was impossible to monitor weight loss or obtain a

representative sample for molecular weight analysis. Therefore, the only samples evaluated were

those subjected to 0. IN NaOH.









Immersion of PLA in Acid Media

Rectangular sheets (6cm x 4cm x 0.75mm) obtained from PLA drinking cup bottoms

(Figure 2-1) were placed in 100ml of aqueous solution of nitric acid at 0.1% or 0.016N (pH 1),

and stored for 22 days at 250C. At the beginning and end of the experiment, structural integrity

and weight loss were assessed. Change in molecular weight was not evaluated because results of

previous analyses proved that PLA samples were not sensitive to acids.

Exposure of PLA to Steam

Rectangular sheets (6cm x 4cm x 0.75mm) obtained from PLA cup bottoms (Figure 2-1)

were prepared and placed inside jars. Lids were adapted with two holes (about 1cm diameter) to

allow steam transfer. Jars were placed in a vertical still retort where steam was fed and

temperature/pressure was controlled with a pneumatic system. Experiments were run at 100, 110

and 1200C, for 1, 2, 3, 4 and 8 hours. After each treatment, samples were quickly cooled in air to

room temperature and dried in an oven at 1050C until constant weight. Structural integrity and

molecular weight of steam-treated PLA samples were evaluated.

Results and Discussion

Exposure of PLA to Gamma Irradiation

Mechanical properties

PLA made from pure L-Lactide, also called poly(L-lactide), is semi-crystalline.

Incorporation or co-polymerization with isomers M-lactide or D-lactide decreases degree of

crystallinity, causing polymers to become more amorphous [24, 25]. PLA resins can be tailor-

made for different fabrication processes, including injection molding, sheet extrusion, blow

molding, thermoforming, film forming, or fiber spinning. Critical factors include degree of

branching, D-isomer content, and molecular weight distribution. For thermoforming, D-isomer









content might be in the range of 4-8% [25]. Unirradiated PLA mechanical properties have been

studied by others and comparisons to oriented polystyrene (OPS) have been made [26].

Rectangular PLA samples showed increasing brittleness with absorbed dose. At higher

doses, samples were sensitive to even careful handling. Results for stress and strain at break

(rupture strength) using the Instron 4301 are shown in Figure 2-3 and Figure 2-4. Higher gamma

irradiation doses reduced PLA mechanical properties in a manner that is similar to traditional

thermoplastic polymers including polystyrene, polypropylene and polyurethane [27, 28, 29].

Other authors studied effects of electron beam irradiation on lactides and found similar

trends [12, 30]. Stress at break of PLA samples irradiated at 172 kGy dropped from 127 MPa to

18MPa, a factor of about 7. For the same samples, strain at break dropped from 75% to 2%.

These numerical values represent a marked increase in brittleness of the irradiated PLA samples.

This behavior suggests crystalline damage due to free radical attack [12].

Molecular weight

Figure 2-5 shows weight-average molecular weight, 1 f,, and viscosity-average molecular

weight, My, for un-irradiated and y-irradiated PLA samples. Since intrinsic viscosities are lower

at higher absorbed doses, molecular weights also conform to the Mark-Houwink equation. This

result confirms that chain scission was the predominant effect of tested irradiation treatments.

Weight-average molecular weight dropped 86% from 16.3 x 104 to 2.3 x 104. Number-average

molecular weight dropped 85% from 15.1 x 104 to 2.2x 104. These molecular weight reductions

were more severe than those found by others for poly(L-lactic) acid [11]. In this work, the

material was commercial thermoformed PLA that includes D-isomers, and this may be the

reason for such difference.









Ratios of weight average to viscosity average molecular weight, Mw/Mv, were 1.08, 1.05

and 1.03 for absorbed doses of 0, 72 and 172 kGy, respectively. These narrow molecular weight

distributions tend to suggest that PLA used for these samples was created from a ring-opening

polymerization process. This happens to be the process claimed by the PLA manufacturer [25].

Degree of chain scission

The plot of 1/.1 1/.\ vs. dose is shown in Figure 2-6. The straight line suggests that

chain scission was random [31]. Chain scission yield, Gs, was found to be 2.21, which is greater

than values found by other researchers for gamma irradiated poly (L-lactic) acid [11], e-beam

irradiated poly (lactide-co-glycolide) and e-beam irradiated poly (L-lactide) [11, 12]. This

suggests that gamma irradiation has a higher effect on chain scission than e-beam irradiation, and

that inclusion of D-isomers in the polymer structure increase such sensitivity.

Exposure of PLA to Electron Beam Irradiation

Before composting

Structural integrity

Figure 2-7 shows digital pictures at magnifications of 10X of irradiated PLA surface. Mainly,

four phenomena occurred after e-beam exposure: (a) twisting, (b) change of color, (c) voids

formation and, (d) structural weaknesses. Also, it was observed that these changes were more

severe as irradiation doses increased.

Twisting may be explained by the relatively high irradiation dose rate resulting in

temperature increase and associated relaxation of polymer chains. The glass transition

temperature for PLA has been reported to be around 580C [10]. Sample color changed from

transparent to yellow, however, transparency returned after about one week. Irradiated polymers

form unstable chromatic groups, possibly by the introduction of conjugation to the carbonyl

groups of the chain [32]. It is well known that radiation induced chemistry continues after









exposure and this is a reason that plastic radiation dosimeters require at least 48 hours before

reaching final significant changes.

The phenomenon related with voids formation is common in abused irradiated materials.

Generally, there should be void nuclei formation followed by swelling, which is attributed to an

increase of temperature and irradiation dose [33]. Irradiated PLA samples showed internal

swelling voids that increased in number and size as irradiation dose increased. Void formation is

likely the result of a combination of chain scission, gas formation, swelling, and associated

polymer rearrangement due to heating.

Polylactic acid samples became extremely brittle, suggesting a reduction in chain length

with irradiation dose. This behavior also suggests damage to crystalline regions due to free

radical attack [12].

Molecular weight

Figure 2-8 shows how PLA molecular weight was affected by e-beam irradiation. PLA

samples treated with 0, 72, 144 and 216 kGy of e-beam irradiation achieved weight-average

molecular weights of 1.7x105, 8.2x104, 6.9x104 and 6.1x104 g/mol. These values represent about

48%, 40% and 35% of the initial molecular weights.

Comparing with results for gamma irradiation (Mw at 172 kGy = 14% of untreated Mw), it

can be said that PLA is more vulnerable to gamma rather than e-beam irradiation. For any of

these treatments, decreases in molecular weight occur due to scission of the main backbone [12].

Degree of chain scission, Gs

The plot of 1/.1 1/1 ., vs. dose, D, is shown in Figure 2-9. The degree of chain scission,

Gs, was found to be 0.52, which is lower than the 2.21 value obtained for gamma irradiation

using Cs-137. It is likely that higher dose rates involved with electron beam irradiation









effectively reduced local oxygen concentrations, which helped to protect polymer samples during

irradiation.

After composting

After 2 weeks of composting, most organic feedstock biomass excluding PLA looked and

smelled as finished compost. Literature indicates that this aerobic bioprocess releases water

vapor (0.6-0.8 g/g), carbon dioxide and heat (about 25 KJ/g), which cause the compost

temperature to rise significantly [20]. Figure 2-10 shows that compost temperatures quickly rose

to about 680C during the first week, fell to 450C by the second week, and leveled off at the

chamber controlled temperature of 350C by the third week.

Samples of PLA after 6 weeks are shown in Figure 2-11. While breakdown was most

severe in highest irradiation dose samples, none of the samples were completely mineralized.

Structural integrity

Figure 2-12 shows views of irradiated/composted PLA and reveals effectiveness of

irradiation as a pre-composting step for enhancing PLA breakdown. Structural integrity was

affected and samples turned from clear to milky, smooth to porous, and glassy to powdery. The

effect was more intense as irradiation dose increased. Samples turned very brittle, showing

sensitivity to even careful handling.

During the composting process, PLA samples are subjected to high relative humidity and

temperature. These conditions are favorable for hydrolysis, where a reorganization of the smaller

chain molecules causes an increase of the polymer crystallinity and opacity [34]. In addition,

lower molecular weight polymers are more hydrophilic than corresponding high molecular

weight polymers because of higher concentrations of hydrophilic end groups (both hydroxyl and

carboxyl). Hydrophilicity affects osmotic pressure causing influx of water into the polymer

matrix, which causes a buildup of hydrostatic pressure and consequent cracking and formation of









microcavities. Microcacavitation occurs due to leaching of PLA material into the surrounding

medium [35].

As long as hydrolysis produces small molecules of PLA, microbial activity will

commence. Microorganisms begin to assimilate small molecules of PLA releasing carbon

dioxide and water vapor, and weakening carbon to carbon bonds. It is presumable that the

combination of these hydrolytic and biological effects is responsible for changes in structural

integrity of PLA, turning it from glassy to porous and finally to powder.

Weight loss

Figure 2-13 shows results of weight loss of irradiated/composted PLA over time.

Reduction of weight suggests metabolic conversion of polymer in compost or into a soluble

form. Greater irradiation doses and compost times resulted in increasing weight loss. For

instance, un-irradiated PLA samples reduced weight by 1.3% after 10 weeks of composting

whereas samples with absorbed doses of 216 kGy had reduced weight by 9.4%.

This study exposed a critical aspect of PLA breakdown and compost behavior. Essentially,

PLA requires relatively warm temperatures in order to soften and open the polymer structure to

biological attack. As biological activity quickly progressed, a lot of heat was released in a

relatively short time. This time was too short to sufficiently convert PLA. Converters and

manufacturers of PLA claim that biodegradation of the polymer takes place only at 580C for

more than 6 weeks. Here, temperature was variable and below 580C after the second week.

After a few weeks in the composer, PLA samples with absorbed doses of 144 and 216

kGy turned very brittle and began to disintegrate from mechanical rotation of the composer. It

is likely that these smaller pieces, with larger surface area to volume ratios, and lower molecular

weights due to irradiation, provided some advantage even as microbial activity diminished over

time.









Molecular weight

Figure 2-14 shows how molecular weight of e-beam irradiated PLA was affected over

composting time. Final weight-average molecular weights of irradiated/composted PLA samples

were 3.55x104, 1.86x104, 1.25x104 and 1.06x104 g/mol for the absorbed doses of 0, 72, 144 and

216 kGy, respectively. These values represent 21%, 11%, 7% and 6% of initial un-irradiated

PLA molecular weight. Therefore, even when breakdown was not totally achieved, it was

demonstrated that e-beam irradiation is effective in improving degradation of PLA during

subsequent composting.

During the composting process, hydrolysis of polymers leads to molecular fragmentation,

which can be regarded as a reverse poly-condensation. This process starts with a water uptake

phase followed by a splitting of ester bonds in a random way according to the Flory principle

[33]. High relative humidity and temperature provide conditions for cleavage of the ester

linkages by water uptake and successive reduction in molecular weight [3, 36], as illustrated in

Figure 2-15.

After hydrolysis, microorganisms assimilate lactic acid oligomers that may be soluble,

releasing carbon dioxide and water. This two-step process demands reduction in PLA molecular

weight early in the composting process. Nevertheless, rate of molecular weight reduction turned

slower after the second week due to a reduction of microbial activity as evidenced by low

temperature (350C instead of recommended 580C) and depletion of nutrients [20].

D-value

Figure 2-16 shows D-value plots for molecular weight versus dose after 0, 1, 2 and 6

weeks in the composer. Associated linear regression data and D-values are summarized in Table

2-1. Good linearity is observed in all plots with r-squares above 0.93.









The D-values of irradiated PLA were about 430, 560, 380 and 410 kGy for 0, 1, 2 and 6

weeks of composting. Initial D-value suggests that e-beam irradiation treatment of 430 kGy on

PLA will reduce its molecular weight in 90%. The apparent increase in D-value (lower apparent

sensitivity to irradiation) during the first week in compost may be due to chain recombination as

compost temperatures increased above Tg. After the first week, D-values take their initial values,

probably due to lower temperature.

These results suggest that the primary effect of electron beam treatments on PLA

composting behavior is the initial reduction of molecular weight. Irradiation essentially provides

a head-start on PLA breakdown, but it does not appear to significantly increase sensitivity of

PLA to hydrolysis during composting.

Immersion of PLA in Alkaline Media

Structural integrity

Over time, it was noticed that PLA samples subjected to 1N NaOH fragmented but without

losing tensile properties. Samples remained hard and brittleness was fairly consistent. For both

conditions (0. IN NaOH and IN NaOH), edges of samples were observed to become rougher

with concentration and time. Figure 2-17 shows magnified pictures (xlO) to illustrate this

phenomenon.

Weight loss

Samples of PLA immersed in IN NaOH fragmented over 22 days to such an extent that

monitoring weight loss was not possible. For PLA samples immersed in 0.1N NaOH for 22 days,

weight loss was around 14% as shown in Figure 2-18.

Since brittleness was not affected, but weight dropped, it is believed PLA dissolves under

alkaline conditions, which is not the same as hydrolysis. Polymer dissolution is a consequence of

molecular disentanglements and occurs more intensely on the surface than internally due to the









direct contact with solvents in these areas. That phenomenon caused a heterogeneous sample in

terms of entanglements.

Molecular weight

Exposure of PLA to alkaline media for 22 days did not significantly affect weight-average

molecular weight (Figure 2-19). Presence of peaks ranging between 1.38x105 and 1.66x105

g/mol may be attributed to sample heterogeneity in terms of entanglements, which occur more at

the surface rather than in the center. Thus, it may be possible that test samples were more

representative of surface material, or vice versa, and affected the numerical results. It is

important to recall that an assumption of the intrinsic viscosity method for molecular weight

estimation is that entanglements are the same in the whole sample [37]. It appears that this

assumption may not be valid in this case.

Immersion of PLA in Acid Media

Structural integrity

Structure was not affected by a solution of nitric acid 0.1% or 0.016N (pH=l) after 22

days. There was no change in color, toughness, brittleness, weakness, or any other phenomena.

This observation suggests that PLA is not vulnerable to acids at a temperature of 250C.

Weight loss

Dry weights of PLA samples did not change after 22 days. Absence of weight loss means

that dissolution or reaction of PLA did not occur in acid media at 250C.

Exposure of PLA to Steam

Structural integrity

Samples of PLA exposed to steam for 4 and 8 hours displayed shrinking, brittleness and

pore formation (Figure 2-20). These changes tended to be more severe as steam temperature and

time increased.









Shrinking is attributed to application of temperatures above the glass transition temperature

(Tg=58-70C) for PLA [10]. The glass transition is a second-order thermodynamic transition

where polymers turn from glassy to rubbery state. In this state, change of volume with

temperature is intensified and is revealed through shrinking and twisting observed in samples.

The mechanism of this phenomenon is translational motion of molecules and cooperative

wriggling and jumping of segments of molecules, leading to flexibility and elasticity [37].

Increased brittleness was quite noticeable, and samples more intensely treated were more

sensitive to subsequent handling. Samples generally exhibited cracks and broke easily. This

suggests formation of amorphous regions caused by smaller molecules, rearrangement and rapid

cooling.

Pores formed during treatment are shown in Figure 2-21. This demonstrates severe

disruption of PLA structure as well as exposure of greater areas of polymer that can be attacked

during subsequent composting. The mechanism of pore formation was not determined, but is

likely to be related to regional leaching of PLA to the surrounding medium as a result of

hydrolysis, leaving cavities or pores.

Molecular weight

Molecular weight analysis was conducted for PLA samples exposed up to 4 hours in

steam. Beyond this time, molecular weight distributions were too broad for analysis. Figure 2-22

shows how PLA molecular weight was affected by steam treatments. Initial weight-average

molecular weight was about 2. 10x105 g/mol. After 4 hours at 100, 110 and 1200C, samples

achieved weight-average molecular weights of 6.00x104, 2.88x104 and 1.19x104 g/mol,

respectively. These values represent about 29%, 14% and 6% of the initial molecular weight, for

each respective treatment.









Data reported by other authors show that polyamide 11 subjected to high temperatures in

acidified water (pH 4) reduces molecular weight by half in about 40 days at 1000C, and 15 days

at 1200C [38]. Results here show that PLA exposed to similar treatments reduces molecular

weight by half in about 2.2 and 0.5 hours, at each respective temperature. While this comparison

indicates that PLA is a good candidate for steam hydrolysis, it also suggests that steam

treatments may also serve as a means to separate PLA from other plastics in the waste stream.

Dramatic decreases in molecular weight are the result of hydrolysis caused by high

temperature and relative humidity, and can be regarded as a reverse poly-condensation. Splitting

of PLA ester bonds requires water and is helped with temperature, occurring in a random way

according to the Flory principle [34]. More severe treatments would involve more energy with

sufficient moisture, yielding higher chain scission, and therefore, lower final molecular weights.

The degree of chain scission was not determined for steam treatment, since crosslinking does not

take place.

Conclusions

After evaluating different treatments to reduce PLA molecular weight, it was concluded

that exposure to steam is most rapid and effective. Samples of PLA treated for 4 hours with

steam at 1200C became extremely brittle and steam caused weight-average molecular weight to

decline by 94%. The mechanism of steam-treated PLA molecular weight reduction is basically

thermal hydrolysis.

Gamma irradiation is the second ranked treatment regarding PLA molecular weight

reduction. Samples treated with 172 kGy y-irradiation decreased molecular weight by 86%. Here,

the main mechanism for this reduction is radiolytic chain scission. Using electron beam

irradiation, results were less satisfactory, which is likely due to protection from oxygen. In an











additional experiment, e-beam irradiated PLA samples showed more sensitivity to composting

conditions than untreated PLA samples, but even so, they did not achieve complete

biodegradation.

Use of alkaline and acid solutions to enhance PLA hydrolysis was not successful at the low

temperatures applied. Alkaline solutions promoted polymer dissolution, which might prove

useful as part of a combined approach for accelerating PLA degradation.

Table 2-2 summarizes molecular weight reduction results for each treatment. Given

availability, familiarity and effectiveness of steam treatment it is likely that steam would be the

treatment of choice for accelerating PLA degradation.


Front View Top View
9Srmm
116 9mm


m62mmm




62mm
Figure 2-1. Drinking cups made of PLA


" T ... :i"


(tcncenlralion) viscosity) (reduced viscosity} (inlrinsic viscosity) ({nolecular weight)
Ct 11 ------ Pr1l -I
C ----- 2 -*P I)re2
C3 ---- --- kr3 [L------- ] M=-k- a

Cn ------ lFr tI


Pred


SC1,, 1


F

Figure 2-2. Principle of intrinsic viscosity method for determination of molecular weight














160


140 -


120 -









60
S100
80 -


60 -


40 -


20 -


0
0 20 40 60 80 100 120 140 160 180 200

Dose (kGy)
Figure 2-3. Stress at break of y-irradiated PLA samples





100 -



80 -



60 -



S 40
"co


% 20



0


0 20 40 60 80 100 120 140 160 180 200

Dose (kGy)
Figure 2-4. Strain at break of y-irradiated PLA samples














2e+5

2e+5

2e+5 -

1e+5

S1e+5
o
le+5

8e+4

6e+4

4e+4

2e+4

0
0 20 40 60 80 100 120 140 160 180 200

Dose (kGy)

Figure 2-5. Molecular weight of y-irradiated PLA samples: Mw, OM,






5e-5 -



4e-5



- 3e-5


0
2e-5



1e-5



0




0 20 40 60 80 100 120 140 160 180 200

Dose (kGy)

Figure 2-6. Determination of Gs in PLA y-irradiation: Gs = 2.21






































Figure 2-7. E-beam irradiated PLA showing voids formation: (a) 0 kGy, (b) 72 kGy, (c) 144
kGy, (d) 216 kGy


1.8e+5


1.6e+5 -


1.4e+5


1.2e+5 -


1.0e+5


8.0e+4 -


6.0e+4


4.0e+4


2.0e+4


0 50 100 150 200

Dose (kGy)
Figure 2-8. Molecular weight of e-beam irradiated PLA: Mw, OM,


I I I I I














1.8e-5

1.6e-5

1.4e-5

1.2e-5

1.0e-5

8.0e-6

6.0e-6

4.0e-6

2.0e-6

0.0


0 50 100 150 200 250

Dose (kGy)
Figure 2-9. Determination of G, in PLA e-beam irradiation: Gs = 0.52



70


Figure 2-10.

Figure 2-10.


10 20 30 40
Days

Temperature profile of compost bulk


50 60 70 80





























Figure 2-11. E-beam irradiated PLA after 6 weeks in compost: (a) 0 kGy, (b) 72 kGy, (c) 144
kGy, (d) 216kGy


Figure 2-12. Structural integrity of e-beam irradiated/composted PLA: (a) early cracks affecting
surface, (b) layer formation and overall structure affected

















8 ....
-e-216 kGy



6
5.7



4
3.3


2
1.3


0
0 2 4 6 8 10 12

Weeks




Figure 2-13. Weight loss of e-beam irradiated/composted PLA


200000





150000


100000


50000





0


0 1 2 3 4 5 6

Weeks


Figure 2-14. Molecular weight of e-beam irradiated/composted PLA












H-,H water


CH3 0 CH3 0 CH,
I || I II I
HO-CH-C--CH-CH-CCH-C---CH- -H-C-OH --
II c II I II
0 CH, 0 CH, 0
n m


CH 0 CH3


I I II
HO-CH-C O-CH-C- O -CH-C-OH

0 CH, O
n
Figure 2-15. Hydrolysis of PLA.


5.5




5.0 -




S4.5-
0)
0
.--


4.0




3.5


0 CH3
II I
+ H-- -CH-C- O-CH-C-OH
SC II
CH m 0
m


Dose(kGy)

Figure 2-16. Plots of e-beam irradiation dose vs. log Mw for D-values estimation


00 wk
SEl wk
A2 wk
X6 wk




.. ..
.-. --. ....... -. .........
-2
...... .......... .





































Figure 2-17. Structural integrity of PLA subjected to alkaline media: (a) 0.1N 2d, (b) 0.1N 22d,
(c) lN 6d, (d) IN 13d


.2 8


6


4


2


0
0 5 10 15 20 25
Days

Figure 2-18. Weight loss of PLA subjected to 0. IN NaOH












180000


150000


120000







90000


Time(d)

Figure 2-19. Molecular weight of PLA subjected to alkaline media


Figure 2-20. Structural integrity of steam-treated PLA: (a)1000C-4h, (b)1100C-4h, (c)1000C-8h,
(d)1100C-8h


0



0


0





































Figure 2-21. Pores in steam-treated PLA: 1200C-8h (x60)


S_100C
T("C) M, drop
100 71% 0 110
--" 110 86%
120 94% A120C







-.-

- -


I I I I


Time (h)
Figure 2-22. Molecular weight of steam-treated PLA


250000




200000




150000


100000 4


50000




0









Table 2-1. Linear regression outputs and D-values of e-beam irradiated/composted PLA
0 wk 1 wk 2 wk 6 wk
R-square 0.9322 0.9481 0.9426 0.9328
Slope -0.0023 -0.0018 -0.0026 -0.0024
D-value 427 564 384 412

Table 2-2. Comparison of treatments to reduce PLA molecular weight
Treatment Mw reduction Conditions Availability (max 5)
Gamma irradiation 86+2% 172 kGy +
E-beam irradiation 65+9% 216 kGy +
Alkaline hydrolysis <10% NaOH 0.1N x 22d +++++
Acid hydrolysis NA HN03 0.1% x 22d +++++
Steam 94% 120C x 4h +++









CHAPTER 3
KINETICS OF REDUCTION OF MOLECULAR WEIGHT IN STEAM-TREATED PLA

Introduction

In the previous chapter, it was demonstrated that exposure of PLA to steam is the most

effective treatment to reduce molecular weight. This chapter covers kinetics of reduction of

molecular weight in steam-treated PLA. A first-order reaction model was proposed. This

mathematical estimation would allow relating molecular weight with steam temperature and

exposure time.

In addition, this chapter confirms that steam affects PLA through hydrolysis. Analysis of

spectroscopy by Fourier Transform Infrared Attenuated Total Reflectance (FTIR-ATR)

confirms depolymerization back to lactic acid.

Methods

First Order Reaction Model

Molecular weight loss of PLA treated with steam at 100, 110 and 1200C was assessed

using first order kinetics. The mathematical model follows Equation 3-1 where kT is the reaction

rate constant at temperature T, and Mis the molecular weight at time, t.

dM
= -kM (3-1)
dT

Solving the ordinary differential equation with limits t = 0 to t, and M Mo to M, results in

Equation 3-2:

M = Moe kT (3-2)

Kinetic constants, kT, were found to be correlated with absolute steam temperatures, T, in

accordance to the Arrhenius behavior [39] shown in Equation 3-3. Activation energy, Ea, and

pre-exponential factor, ko, were estimated by linearization.










k, = ko exp(- (3-3)
RT

A final model for molecular weight of PLA, which is temperature and time dependent, is

shown in Equation 3-4. This model allows prediction of molecular weight, M, of PLA with initial

molecular weight, Mo, after exposure to steam at temperature, T, after time, t.

M = Mo exp(-kt)= Mo exp(-kot exp(- )) (3-4)

Depolymerization of PLA to Lactic Acid Resulting from Steam Exposure

An experiment was conducted in order to confirm that exposure of PLA to steam

undergoes hydrolysis until complete depolymerization to lactic acid units. For this purpose, an

extreme treatment of 1200C for 24 hours was performed. In this experiment, PLA samples

(-30. g) were placed in jars (holed lids) containing 100ml of deionized water (pH 7.5) and then

retorted.

After steam treatment, jars contained residual solid PLA and a liquid drip, probably

condensed water plus a byproduct from PLA. Drip was collected and analyzed by pH

(Accumet AR60, Fischer Scientific, Pittsburgh, PA) and FTIR-ATR (Nicomet 6700 Smart

Orbit, Thermo Scientific, Inc) to confirm presence of lactic acid. Also, the yield of PLA

conversion to its monomer lactic acid was determined (Equation 3-5). Variables Wo and Wfare

PLA dry solid weights before and after the steam exposure for 24 hours. The term (Wo Wf)

represents the weight of PLA that was converted to lactic acid.

W -W
% conversion= x 100% (3-5)
wel









Results and Discussion


First-order Reaction Model

Figure 3-1 shows semi-log plots of molecular weight vs. time at 100, 110 and 1200C.

Slopes of curves, obtained by regression analysis, represent the respective kinetic constants, kT,

and are shown in Table 3-1.

Figure 3-2 shows temperature sensitivity of the reaction rate constants, kT, through the

Arrhenius plot. Regression provides estimates for activation energy, Ea, and Arrhenius pre-

exponential factor, ko. Regression analysis yields an r-square of 0.996. Values for Ea and ko were

52.3 KJ/mol and -6.18x106 h-1. Other authors have found Ea of 51 KJ/mol for poly(L-lactic acid)

(PLLA) in the melt at 180-2500C [39] and 233 KJ/mol for PET in the melt at 250-2800C [40].

This shows that activation energy for reduction of PLA molecular weight is much lower than that

of PET, but similar to that for PLLA in a melt at higher temperatures. These values were used in

Equation 3-4 to predict molecular weight reductions shown earlier in Figure 2-22. Figure 2-22

shows experimental and model predicted values.

Depolymeryzation of PLA to Lactic Acid Resulting from Steam Exposure

After exposure of PLA samples to steam at 1200C for 24 hours, the solid mass was

significantly reduced. This reduction suggested PLA conversion to a water-soluble compound in

solution with condensed steam. Liquid in the jar (drip) had a pH of about 1.5 indicating presence

of acid, most probably lactic acid. Finally, results of FTIR-ATR confirmed that the liquid by-

product was lactic acid. Figure 3-3 shows spectra for lactic acid from PLA and of DL-lactic acid

control (Acros Organics, Geel, Belgium). These spectra appear to confirm liberation of lactic

acid during steam treatment.

As seen in the mass balance shown in Figure 3-4, weight of PLA decreased from 30. Ig to

4.6g after this treatment. This represents a percentage of weight loss of 84.7% where a









significant part converted to lactic acid units. Tsuji et al. (2003) studied melt hydrolysis of PLA

and found a yield of L-lactic acid from PLLA of 90% at 2500C for 10-20 minutes [39]. Ohkita

and Lee (2006) investigated the enzymatic hydrolysis of PLA using proteinase K and found a

yield of lactic acid from PLA of 38% after 8 days at 370C [8]. Based on weight loss of PLA, it is

presumable that yields of lactic acid here were not much different from those observed by

hydrolysis in the melt.

Conclusion

It has been demonstrated that exposure to steam up to 1200C is an excellent method to

hydrolyze PLA. Treated samples became brittle and riddled with pores, and analysis performed

by FTIR-ATR confirmed hydrolytic liberation of lactic acid to the treatment medium. Weight

loss of PLA after 24 hours in steam at 1200C was 84.7% and suggests significant conversion to

lactic acid.

A kinetic model describing molecular weight reduction in PLA as a consequence of

hydrolytic steam treatments was presented. Degradation followed first order kinetics with

activation energy, Ea, of 52.3 KJ/mol. Predicted values fitted experimental data well.

Availability of this model is very important because, if it is demonstrated that steam-treated PLA

is affected under composting conditions, additional models may be developed to predict

biodegradation behavior as a function of the steam-treatment conditions. These simulations will

ultimately be useful for optimizing the whole two-step process (pre-treatment and composting).

Finally, this study shows that steam treatments may be suitable not only for making PLA

waste more accommodating to commercial composting operations, but also, for assisting with

separation of PLA from traditional plastic wastes.



































0 1 2 3 4

Time (h)

Figure 3-1. First-order reaction plots for steam-treated PLA


2.50E-03
0.00 -


2.55E-03


2.60E-03


2.65E-03


-0.50 --


-1.00 +


-1.50


1/T ( K-1)
Figure 3-2. Arrhenius plot for steam-treated PLA


2.70E-03


Ea 52.3 KJ/mol
Ko =-6177171 h-1












Library: UF PKG lab
DL-Lactic acid


Lactic acid obtained
from PLA


4000 3500 3000 2500 2000 1500 1000 50C
Wavenumbers (cm-1)
Figure 3-3. Spectra of lactic acid obtained by FTIR-ATR


Steam )


PLA (30. g) + water (100g)



Steam exposure 1200C x 24h /
condensation


Steam + water vapor


PLA wet + water +
lactic acid


Drip (101.9g)







Water vapor


PLA (4.6g)

Figure 3-4. Mass balance of PLA treated with steam at extreme conditions


PLA wet


t









Table 3-1. Reaction rate constants, k (h-1), for molecular weight reductions of steam-treated PLA
Temperature (OC)
100 110 120
k 0.290 0.470 0.680
r2 0.975 0.989 0.988









CHAPTER 4
DEVELOPMENT OF METHODS TO EVALUATE AEROBIC BIODEGRADATION OF PLA

Introduction

The following standards are available for determining aerobic biodegradation of plastic

materials under controlled composting conditions:

American Society for Testing and Materials: ASTM D5338
International Standards Organization: ISO 14855

These methods require mounting complicated systems involving temperature controlled

vessels connected to permanent supply of oxygen and water vapor. Proposed vessels are too big

(2-5 L) and temperature profiles are generated, providing variability in conditions inside the

bulk. Additionally, heat generation can vary depending on compost weight and composition,

which may also contribute to uneven temperature profiles. Figure 4-1 shows a diagram of the

system configuration for ASTM D5338 [40], and it is required oxygen tanks, humidifiers, gas

chromatographs, and baths / big ovens/ heaters. These issues, combined with monitoring duties,

makes this method not practical for rapid biodegradation assessments. A simpler approach would

be desirable. This chapter investigates new ways to determine evolution of aerobic

biodegradation instead of using standard methods.

Aerobic biodegradation of PLA is measured by the ratio of carbon evolved as carbon

dioxide (released during breakdown), over initial carbon content. Mathematically, it can be

expressed as Equation 4-1.

Mass of carbon in evolved CO2 10 (
% biodegradation = x 100% (4-1)
Mass of carbon in initial PLA

If none of the carbon atoms in PLA molecules converts to carbon in the generated CO2,

biodegradation would be zero. In contrast, if all carbon is converted to C02, then biodegradation

would be 100%. Some carbon is believed to be converted to bicarbonates and carbonic acid, and









other components may recombine with the humic acids present in the compost. Also, carbon

from PLA may be assimilated into microbial biomass. However, production of CO2 is the basis

of biodegradation assessments. Therefore, the capability of quantifying evolved CO2 from the

system is critical to any method developed to quantify biodegradation.

In this chapter, three methods are designed, developed and assessed to measure PLA

biodegradation evolution, including (1) the method of flexible bags, (2) the method of rigid

containers, and (3) the method of perforated jars. The first two rely on principles of modified

atmosphere packaging (MAP) and estimate CO2 production by combining a system molar

balance and equations that govern gas permeability through plastic films. The third method relies

on principles of gas diffusion and estimate CO2 production by combining a system molar balance

and CO2 diffusion through holes in jars.

Materials and Methods

Method of Flexible Bags

This method was proposed as a modification of the standard method ASTM D5338 [41] to

overcome the issue of requiring permanent oxygen and water vapor supply. The use of a highly

permeable film would permit oxygen transmission into the bag while withholding moisture

content of the biomass. Thus, selection of the film is critical for this method. Ideally, this method

requires a film that is very permeable to oxygen, impermeable to water vapor, and semi-

permeable to carbon dioxide. Unfortunately, no commercially available films meet these

requirements, however high oxygen transmission rate (OTR) polyethylene was the closest to the

requirements of this method, so it was used.

Polyethylene film (Cryovac, Duncan SC) of 3.035mil thickness with high oxygen

transmission rate (9224 cc m-2d1 @ 250C) was used to prepare bags with dimensions 9cm x

12cm. A thermosealer (Sencorp Systems model 12SC/1) was used for sealing edges. Rubber









pads (sticky nickels, Mocon, Inc., Minneapolis, MN) were glued on their surfaces to serve as

syringe needle ports injecting water or taking gas samples from the headspace. Bags were filled

with 40g of 3-month mature compost (Appendix A) and 8g of PLA. Control bags included only

40 g of compost. Filled and sealed bags were stored in a Lab-Line L-C Incubator (Lab-Line

Instruments, Inc., Melrose Park, IL) at 580C for 40 days. Figure 4-2 shows a diagram of the

flexible bags and the main interactions between the biomass (compost + PLA), the headspace

and the environment, and pictures can be seen in Figure 4-3.

Periodically, bags were agitated, water was injected (-1-3ml) and gas samples from the

headspace were taken for analysis. Headspace gas analysis was performed using a dual

headspace analyzer (Pac Check 650, Mocon, Inc., Minneapolis, MN) to determine carbon

dioxide and oxygen concentration over time. A molar balance of carbon dioxide in the system is

expressed in Equation 4-2. Oxygen and nitrogen concentrations were not used for this method

since they are not a carbon source.

qr CO2 generated = r CO2 permeated + r CO2 headspace (4-2)


The number of moles permeated can be estimated by Equation 4-3, derived from the

definition of permeability [42].

PCO2 A pco2 At PCO2 A [CO2 aptm t
q CO2 permeated = (4-3)
E E

The number of moles in the headspace can be estimated by Equation 4-4, derived from the

universal gas law [43].


7 CO2 headspace = O2 hs t P (4-4)
RT RT









Film permeability to carbon dioxide, Pco2, at 580C was estimated using the methodology

described in Appendix B and was equal to 13.41 mol.mil / (atm.d.m2). The volume of the

headspace, Vhs, was estimated by subtracting the volume occupied by the compacted biomass

(Appendix C) to the bag volume (Appendix D) which was measured with a volume-meter

designed and constructed for this purpose.

Production of CO2 solely from PLA is the difference between CO2 produced from the

mixture PLA-compost and CO2 produced by the compost itself (control). Once it is known how

many moles of CO2 are due to PLA, the carbon mass can be determined by multiplying the

number of moles by 12 which is the molecular weight of carbon. Finally, total biodegradation is

the ratio of carbon mass evolved as CO2 to initial carbon mass in PLA sample assessed. Weight

of carbon in PLA is half its total weight (Figure 4-4).

To organize and simplify calculations, Table 4-1 was developed. Plots of columns (1) and

(10) provide PLA biodegradation.

Method of Rigid Containers with Plastic Film Lids

A cylindrical, rigid, wide mouth container with 2850cc capacity was adapted with ports,

filled with 100g of 4-month mature compost (Appendix A) and 10g of PLA. Control was filled

with only 100g of compost. Mouths of containers, with internal diameters of 15cm, were covered

with high OTR polyethylene film (Cryovac, Duncan SC) (thickness -3.035mil, OTR -9224

cc/(m2.d) @ 250C) that served as the material to allow 02 and CO2 permeation. Containers were

stored in a Lab-Line L-C Incubator (Lab-Line Instruments, Inc., Melrose Park, IL) at 580C for

18 days. Figure 4-5 shows a diagram of the rigid containers with plastic lids, as well as the main

interactions between biomass (compost + PLA), headspace and environment. A picture of the

system is shown in Figure 4-6.









Daily, containers were agitated, water was injected to maintain proper moisture (-45-

55%), and gas samples from the headspace were taken for analysis. Also, for better performance,

oxygen was injected daily (-100 300cc) to maintain aerobic conditions. The headspace was

analyzed in a dual headspace analyzer (Pac Check 650, Mocon, Inc., Minneapolis, MN) to

determine carbon dioxide and oxygen concentrations.

Over time, data for carbon dioxide concentration in the headspace were collected. As in the

method of flexible bags, the number of moles of CO2 generated by the biomass is the sum of the

moles permeated out of the container and the moles in the headspace. Expressions that govern

each component of this molar balance are Equation 4-3 and Equation 4-4 but with the new data

from this experiment. Thus, Table 4-1 was also used to process these data and plots of columns

(1) and (10) provided evolution of PLA biodegradation assessments.

Method of Perforated Jars

Glass mason jars of 936cc of capacity were filled with 100g of 5-month mature compost

(Appendix A) and 10g of PLA. Controls were filled with only 100g of compost. Lids were

drilled with five 1/16" holes to allow gas transfer between the environment and jars headspace.

Closed jars were stored in a Lab-Line L-C Incubator (Lab-Line Instruments, Inc., Melrose Park,

IL) at 580C for 34 days. Figure 4-7 shows the diagram of perforated jars and the main

interactions between biomass (compost + PLA), headspace and environment. Pictures of a jar

filled with 1-week biomass and arrangement of 5-holes is shown in Figure 4-8.

Daily, jars were agitated and concentration of oxygen and carbon dioxide in the headspace

were determined using a dual headspace analyzer (Pac Check 650, Mocon, Inc., Minneapolis,

MN). Periodically, water (-2-4cc) was added through the holes to compensate for water loss. A

molar balance of carbon dioxide in the system is as follows (Equation 4-5):

q CO2 generated = q CO2 diffused + q CO2 headspace (4-5)









The number of moles, fl, diffused represents the average of moles flowing through the

holes. This value can be estimated by Equation 4-6, derived from Fick's first law that states that

the flux or rate of transport of an ideal gas is linearly related to its concentration gradient [42].

q CO2 diffused = De [CO, ] At (4-6)


The number of moles in the headspace can be estimated by Equation 4-7 which is derived

from the universal gas law [43].


q CO2 headspace = Po2 Vhs Patm [CO2]V, (4-7)
RT RT


The overall coefficient of diffusion, Def, was determined in a parallel experiment

(Appendix E). The volume of the headspace was estimated by subtracting the volume occupied

by the biomass (Appendix C) from the total volume of the jars which was 936cc.

Production of CO2 solely from PLA is the difference between CO2 produced from the

mixture PLA-compost and CO2 produced from the compost itself (control). The carbon mass can

be determined by multiplying the number of moles of CO2 produced by 12, which is the

molecular weight of carbon. Finally, total biodegradation is the ratio of carbon mass evolved as

CO2 to initial carbon mass of PLA samples assessed. Initial weight of carbon in PLA samples is

half the sample weight based on the molecular formula of PLA.

To organize and simplify calculations, Table 4-2 can be completed. Plots of columns (1)

and (10) provide PLA biodegradation.

Results and Discussions

Method of Flexible Bags

The method of flexible bags was developed to simplify analysis of biodegradation

behavior. Ideally, this method could eliminate the need for continuous gas flow and moisture









management. To sustain aerobic composting conditions the film requires high OTR

characteristics. The film chosen has the highest OTR of any readily available commercial film.

At composting conditions, the film proved to be too fragile to manipulate. Additionally, the

film's water vapor transmission rate (WVTR) proved to be higher than desirable for this

application. However, the theory supporting the approach is sound and this analysis and

summary are provided as a means for suggesting future work if and when a better film becomes

available. Main issues encountered during the experiment performed with the method of flexible

bags follow:

Moisture loss and related punctures: Water vapor permeated out of the bag faster than
expected, so moisture content of biomass dropped quickly. This problem was solved by
injecting water into bags more frequently, but this required additional punctures and
resultant leaks.

Abrasion of film: Shaking of the bags, intended to homogenize internal conditions
(oxygen supply, heat and moisture), caused abrasion and damage to the film. This may
have created small holes and leaks that were not visible.

Strength loss of film: Formation of by-products from PLA biodegradation (i.e. lactic
acid) reduced the pH of the biomass, and in concurrence with temperature and high
relative humidity, deteriorated the film surface. Film developed a bur-like appearance
and lost strength. Modification of film properties could affect permeability of the film to
CO2.

Oxygen depletion: When biological activity began to take place, samples started to show
elevated rates of oxygen consumption. This fact depleted the oxygen supply in the
headspace and turned the process anaerobic. ASTM D5338 recommends oxygen levels
above 6% for ensure aerobic conditions [41], and some of the experiments got 0%
oxygen. This issue could have been overcome by increasing the film area. However,
something unexpected was found; even without oxygen, CO2 generation still increased.
This suggests that facultative microorganisms were active and may have been responsible
for biological activity with or without oxygen. Figure 4-9 shows a picture of a bag in its
day 40th that after many days under anaerobic conditions, showed total disappearance of
PLA.

Results of untreated PLA biodegradation, obtained using this method, are shown in Figure

4-10. Large standard deviations were found among the 5 replications performed. For example,

after 40 days one sample was 7.5% biodegraded whereas another was 30.3%. This disparity was









primarily due to issues related to the film used. Thus, this method using currently available high

permeation film is not sufficiently reliable.

Method of Rigid Containers with Plastic Film Lids

The method of rigid containers with plastic film lids was designed to overcome the issues

encountered in the method of flexible bags. The design involved ports attached to the walls of

the rigid container in order to avoid damaging the delicate film. Also, since the plastic film

served as the container's lid (did not contact the biomass), abrasion and strength loss issues were

eliminated. To avoid oxygen and water vapor depletion, gases were injected daily to maintain

required ranges (02 between 10-21%, moisture between 45-60%).

An unavoidable issue was the plastic lid deformation caused by the water vapor pressure,

and its consequent effect in the permeability estimation. Deformation of the plastic film lid was

measured and estimated to add 5%.

After 18 days in rigid containers with plastic film lids, PLA biodegradation was estimated

to be 10.1% as seen in Figure 4-11. As with experiments of the flexible bags, the trend was

almost constant during the first week and then increased at a constant rate. It is believed that in

the presence of PLA, microorganisms slow down biological activity in the surrounding organic

matter (compost) in order to adapt to the new source of carbon.

This method overcomes most of the issues encountered in the method of flexible bags, but

still left room for improvement. Remaining issues are that CO2 permeability may be affected by

water vapor as well as issues with variable geometry due to stretching. These observations led to

a new method that avoided plastic film.

Method of Perforated Jars

The method of perforated jars did not present the issues encountered in previous developed

methods. The problems of area, volume and leaking of the system were eliminated by the rigidity









of the container, and water vapor pressure effects were avoided by presence of holes. The holes

served not only to allow mass transfer (02, C02), but also to inject water or take gas sample from

the headspace. The overall effective coefficient of diffusion (5 holes) for CO2 was found to be

Def= 0.00031 mol/h/% and was estimated by the slope of the plot of experimental calculated

effusion rate vs. %CO2 shown in Figure 4-12. The r-square value obtained by linear regression

analysis was found to be practically 1, indicating an almost perfect linear dependence of effusion

rate with CO2 concentration inside the jar, as expected.

In Figure 4-13, PLA biodegradation was obtained for a remaining solid sample (no drip)

that was exposed to steam at 1000C for 4 hours. Higher rates of biodegradation were observed in

these samples compared with those of untreated samples assessed using the method of rigid

containers with plastic film lids.

Conclusion

Among the methods developed and assessed to perform PLA biodegradation, the method

of perforated jars was found to be the most reliable and simple. It does not require mounting a

system as the one proposed in the standards (ASTM D5338 and ISO 14855) that need permanent

oxygen and water vapor supply. This method is much easier to conduct and adapt. For example,

if it is expected that biological consumption of oxygen will be much faster, more holes could be

added. The only drawback of this method is the requirement to inject water about every two days

to maintain moisture content in the proper range. In this regard, a recommendation could be

integration of a water dispenser to the jars in order to recover water released through holes as

water vapor.

The method of flexible bags and rigid containers with plastic film lids had too many issues,

the most significant being the possible alteration of the film due to high relative humidity,

temperature and vapor pressure in the system. This issue can be overcome by determining a











dynamic permeability coefficient of the film to carbon dioxide, which would be a function of

temperature, and relative humidity and vapor pressure as well. Identifying a film that fulfills

requirements of gas transmission rates (C02, 02 and WVTR) is also difficult at this time.


Figure 4-1. Set up for plastic biodegradation assessment in compost ASTM D5538

Figure 4-1. Set up for plastic biodegradation assessment in compost ASTM D5538


Oven at 580C


O2 permeated


C02 permeated


Water vapor permeate d


Figure 4-2. Interactions in flexible bags



































ctures of flexible bags with


0

--O-CH-C--

CH n


C: 36n g/mol
0: 32n g/mol
H: 4n g/mol
PLA: 72n g/mol
C/PLA: 0.5


Figure 4-4. Chemical formula of PLA










Oven at 58C


02 perm
Film -


CO2 permeated
heated Water vapor permeated

,4_._4


Headspace


O02 generated

t


H20

Jr


Biomass


ort


Rigid container


Figure 4-5. Interactions in rigid container with plastic film lid


t~6 '3-.<


Figure 4-6. Picture of rigid container with plastic film lid








Oven at580C


Perforated lid


CO2 diffused
02 diffused Water vapor diffused

v--! -


02 generated

t


Headspace

2 H20

I I


Biomass


Figure 4-7. Interaction in perforated jar


Figure 4-8. Picture of jar filled with biomass and perforated lid





































Figure 4-9. Picture of compost plus PLA after 40 days, in and out of the bag


40



30-



20
0


10
-II




0-



-10 .
0 10 20 30 40 50

Days
Figure 4-10. Biodegradation of PLA using the method of flexible bags










































Days


Biodegradation of PLA using the method of rigid containers with plastic film lids


0



Figure 4-12.


1 2 3 4 5 6

% CO2

Overall effective coefficient of diffusion for CO2 through 5 holes


O
0

0

0


0

0

0

0
0

0
0

o



0
o

o

O

OO

O'O


02 4 6 8 10 12 14 16 18


2



0



-2


Figure


4-11.


Def= 0.00031 mol/h/%


0.0020


0.0018


0.0016


0.0014


0.0012


0.0010


0.0008


0.0006


0.0004


0.0002


0.0000


I I I I I I












60

50

40

0
m 30
ss


S 2 4 6 8 10 12 14 16 1
-10
Days
Figure 4-13. Biodegradation of PLA using the method of perforated jars


Table 4-1. Biodegradation of PLA by method of flexible bags
(1) (2) (3) (4)
Time, t [CO2] r CO2 r CO2 permeated
permeated accumulated


(5)
r CO2
in headspace


Determined in Use Eq.4-3 (3)t=i + (4)t=i-1 Use eq.4-4
headspace analyzer

Table 4-1. Continued
(6) (7) (8) (9) (10)
r CO2 total r CO2 total r CO2 total Grams of carbon % biodegradation
produced produced by control produced by PLA in CO2 from PLA
(4)+(5) Follow (1) to (6) (6)- (7) 12 (8) (9)*100
for bags with only /0.5*WPLA
compost


Table 4-2. Biodegradation of PLA by the method of perforated jars
(1) (2) (3) (4) (5)
Time, t [CO2] r CO2 diffused r CO2 permeated r CO2
accumulated in headspace
Determined though Use Eq.4-6 (3)t=i + (4)t=i-1 Use eq.4-7
headspace analyzer









Table 4-2. Continued
(6) (7) (8)
r CO2 total r CO2 total r CO2 total
produced produced by control produced by PLA
(4)+ (5) Follow (1) to (6) (6)- (7)
for bags with only
compost


Grams of carbon
in CO2 from PLA
12 (8)


(10)
% biodegradation

(9)*100
/0.5*WPLA









CHAPTER 5
BIODEGRADATION OF TREATED PLA UNDER ANAEROBIC CONDITIONS

Introduction

Although the goal of post-consumer use of PLA is to return to the environment during

composting, this is not currently happening. Separating PLA from waste and acceptance of PLA

in composting plants are proving problematic. Instead, PLA waste is being sent to landfills,

where anaerobic conditions prevail and it appears that PLA does not biodegrade significantly [9].

Little is known about the fate of PLA in landfills. Therefore, an objective of this study was

to evaluate PLA degradation under anaerobic conditions. Under anaerobic conditions, organic

matter usually degrades in four stages: (a) hydrolysis, (b) acidogenesis, (c) acetogenesis, and (c)

methanogenesis [44, 45]. During hydrolysis, molecules split and become smaller and soluble

resulting in conversion of carbohydrates, fats and proteins, into sugars, fatty acids and amino

acids. This chemical reaction requires water and is aided by temperature and enzymes. Later,

during acidogenesis, simpler compounds undergo fermentation carried out by acidogenic

bacteria, and produce volatile fatty acids, hydrogen and carbon dioxide. Later, during

acetogenesis, acetic acid, hydrogen and carbon dioxide are produced. During methanogenesis,

the final products of the anaerobic digestion are obtained. These are methane and carbon dioxide.

For this work, it was postulated that untreated PLA biodegrades at a very slow rate because

of its large molecular weight, making the necessary first step of hydrolysis difficult. However,

pretreatments with steam or irradiation may assists with and/or substitute for hydrolysis and

therefore allow PLA to be converted. It is supposed that main anaerobic reactions for converting

PLA to biogas follow the sequence given in Figure 5-1. It is suspected that the amount of each

intermediate product depends on the microbial strains and temperatures of incubation.









Experiments performed include weight loss of irradiated PLA (gamma and e-beam) and

biochemical methane potential (BMP) of steam-treated PLA (1200C x Oh, 3h), both under

mesophilic and thermophilic conditions. Also, an experiment of untreated PLA in DI water in

absence of oxygen was conducted to assess thermal hydrolytic degradation by itself.

Materials and Methods

Weight loss of PLA in Water under Anoxic Conditions

Rectangular sheets of PLA (1cm x 4cm x 0.2mm) were placed in glass bottles (cap 280ml)

containing 100ml of DI water. Jars were flushed with gas N2/CO2 (70/30) for 30 minutes to

remove oxygen and then immediately sealed. Some jars were stored for 180 days at 370C in an

IsotempTM Low Temperature Incubator (Fisher Scientific, Inc., Philadelphia, PA). Others, at

580C in a Lab-Line L-C Incubator (Lab-Line Instruments, Inc., Melrose Park, IL) for the same

time. Dried and cleaned PLA samples were weighted at the beginning and the end of the

experiment to evaluate their thermal degradation in absence of oxygen at 370C and 580C.

Weight Loss of Irradiated PLA in Anaerobic Biological Media

Clear glass bottles (cap. 280ml) were filled with small pieces of irradiated PLA (-5mm x

7mm x 0.2mm), 90 ml of nutrient formula (Appendix F) and 10 ml of inoculum. Irradiated PLA

pieces were subjected to gamma rays from cesium 137 source (0, 72 and 172 kGy) and electron

beam (0, 72, 144 and 216 kGy). Controls consisted of media and inoculum but without PLA.

Inoculum was provided by the Bioprocess Lab of the Agricultural & Biological Engineering

Department at University of Florida. Inoculum was cultures of naturally occurring

microorganisms, capable of growing under mesophilic (25-450C) or thermophilic (>450C)

conditions [19]. After bottles were filled, they were flushed with gas N2/CO2 (70/30) for 30

minutes to remove oxygen. Bottles were sealed and stored for 180 days at 370C in a IsotempT

Low Temperature Incubator (Fisher Scientific, Inc., Philadelphia, PA), and at 580C in a Lab-









Line L-C Incubator (Lab-Line Instruments, Inc., Melrose Park, IL). These temperatures

provided favorable conditions for biological activity of mesophilic and thermophilic

microorganisms, respectively.

Weight of dried and cleaned PLA samples was determined at the beginning and end of the

experiment to evaluate effects of irradiation in anaerobic biodegradation. Bottles that were set up

looked like in Figure 5-2.

Biochemical Methane Potential (BMP) of Steam-Treated PLA in Anaerobic Biological
Media

The biochemical methane potential 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 substrates.

Clear glass serum-bottles (cap. 280ml) were filled with 1 gram of ground solid steam-

treated PLA, 100 ml of nutrient formula (Appendix F) and 10 ml of naturally occurring inoculum

(mesophilic or thermophilic) supplied by the Bioprocessing Lab of the Agricultural & Biological

Engineering Department at University of Florida. Grinding of PLA was performed using an

Urschel 3600 grinder (Urschel Laboratories, Inc., Valparaiso, IN) with a 3mm screen. Ground

PLA was a sample of the solid residue after steam treatment (1200C x Oh and 3h). Drip was

discarded. Controls consisted of glass bottles with the nutrient formula and inoculum, but

without PLA. After filling, bottles were flushed with gas N2/C02 (70/30) for 30 minutes to

remove oxygen. Later, bottles were sealed and stored for 28 days at 370C in a IsotempTM Low

Temperature Incubator (Fisher Scientific, Inc., Philadelphia, PA), and at 580C in a Lab-Line L-

C Incubator (Lab-Line Instruments, Inc., Melrose Park, IL). These temperatures provided

favorable conditions for biological activity of mesophilic and thermophilic microorganisms.









Biochemical methane potential bottles and steam-treated ground PLA looked as in Figure 5-2

and Figure 5-3.

Weekly, gas production and composition were determined using a Gas Partitioner

Chromatograph model 1200 (Fisher Scientific, Inc., Philadelphia, PA) adapted with a thermal

conductivity detector. Biochemical methane potential (BMP) of PLA was expressed as yield of

methane per gram of PLA sample loaded into BMP bottles, and was determined in accordance to

ASTM El 196 [46]. Data collected were inserted into columns (1), (2) and (3) of Table 5-1. The

volume of methane removed was calculated and expressed at standard conditions of temperature

and pressure (0C, latm) after removing the vapor pressure effect at respective temperatures.

Plots of columns (1) and (11) depict the yield of methane per gram of PLA (untreated and steam-

treated).

Results and Discussion

Weight Loss of PLA in Water Under Anoxic Conditions

Table 5-2 shows the weight loss of untreated PLA in oxygen-free water after 180 days.

Results demonstrate that degradation occurs at 580C but not at 370C. Evaluated samples were not

subjected to any kind of chemical reaction or biological activity, so mechanism for degradation

in absence of oxygen was solely thermal hydrolysis.

Weight Loss of Irradiated PLA in Anaerobic Biological Media

Under thermophilic conditions (580C), all samples were disintegrated and lost in the

media. It can be said that weight loss was almost 100%. In some cases, gelatinous PLA pieces

were found which easily dissolved in the media when subjected to agitation. At this temperature,

the mechanism of PLA degradation is a combination of thermal hydrolysis and biological

activity that would produce methane. These data were not useful for determining effects of









irradiation dose, irradiation source and biological activity on PLA, so data collected from

mesophilic conditions were used for this purpose.

Figure 5-4 shows weight loss of irradiated PLA (gamma and e-beam) under mesophilic

conditions (370C) after 180 days. PLA samples appeared to be much more vulnerable to

anaerobic biological media when pre-irradiated with gamma source rather than e-beam. For

instance, y-irradiated PLA at 172 kGy lost 45% of weight whereas e-beam irradiated PLA at 216

kGy lost only 3% under same anaerobic biological conditions after 180 days. This difference

likely occurred because gamma irradiated PLA samples had lower molecular weights and weaker

structures than those irradiated with electron beam (see Figure 2-5 and Figure 2-8), so they were

more sensitive to biological activity.

At 370C, un-irradiated PLA samples presented negligible weight loss under anaerobic

conditions. But, as seen in Figure 5-4, irradiation dose played an important and accelerated role

(more when treated with gamma rays) in PLA anaerobic biodegradation. That means that at

higher irradiation doses, the weight loss effect was much more pronounced than at lower

irradiation doses. At this temperature, the main mechanism for weight loss is biological activity

performed by mesophilic microorganisms, since thermal hydrolysis has been shown not to occur.

Biochemical Methane Potential (BMP) of Steam-Treated PLA in Anaerobic Biological
Media

Figure 5-5 shows methane production by untreated and steam-treated PLA under anaerobic

conditions. Untreated PLA subjected to mesophilic conditions did not degrade. However,

samples subjected to thermophilic conditions did degrade. Therefore, temperature of incubation

is a key factor for anaerobic biodegradation of PLA. Results obtained for mesophilic conditions

match data from literature [4, 47]. Untreated PLA subjected to thermophilic conditions produced

187 ccCH4/g in 56 days. No data has been found in literature regarding PLA degradation under









thermophilic conditions. In this work, it was demonstrated that PLA evolves methane after 21

days in anaerobic media at 580C. This result was anticipated from prior work with compost when

conditions unintentionally became anaerobic, but yet CO2 production at 580C was observed. For

these samples, hydrolysis and acidification took place mostly during the first 3 weeks, followed

by methanogenesis.

Regardless of temperature of incubation, steam-treated PLA samples (1200C x 3h)

produced more methane than untreated PLA. Under mesophilic and thermophilic conditions,

yields of 90 and 225 ccCH4/g were obtained for treated PLA (improving "negligible" and 187

ccCH4/g from untreated). At 580C, steam-treated PLA began producing CH4 at the beginning of

incubation, whereas for untreated PLA, CH4 was not observed until after the third week. This

suggests that steam treatment provides a head start effect. At 37C, it was shown that PLA can

biodegrade in anaerobic media only if material is pretreated.

Conclusions

Untreated PLA does not degrade under mesophilic conditions, but does under thermophilic

conditions. Methane yield of 187 ccCH4/g after 56 days at 580C was observed. Mechanism for

degradation is hydrolysis and acidification during the first 3 weeks, followed by methanogenesis.

Treated PLA, using steam or irradiation, does biodegrade under anaerobic conditions,

either in mesophilic or thermophilic circumstances. This means that it will ultimately evolve as

carbon dioxide and methane at either temperature range. For steam-treated PLA (1200C x 3h),

methane production was 90 and 225 ccCH4/g after 56 days of incubation at 370C and 580C.

Gamma irradiated PLA samples biodegraded faster than e-beam irradiated PLA, which is related

to oxygen protection afforded by high dose rates associated with e-beam treatments. Gamma

treatment was more effective in reducing molecular weight, which contributed to the head start

effect of y-radiation. Gamma irradiated PLA with absorbed dose of 172 kGy lost 45% of its









weight at 370C after 180 days. This reduction in weight is mainly caused by biological

conversion of PLA to aqueous soluble oligomers, lactic acid, acetic acid, and probably evolution

to carbon dioxide and methane. Also, it was noticed that regardless of irradiation source (i.e

gamma irradiation/Ce 137 or e-beam), higher absorbed doses provided higher rates of

biodegradation.

Treated PLA + H20 o. Oligomers of lactic acid [, i
Hydrolysis
Oli0omers of LA l Lactic acid

Lactic acid Ip Acetic acid + alcohol + CO2 + H20 (Acido/acetok esi

Acetic acid 0 CH4 + CO2 I M.ar-a. n.i

Figure 5-1. Supposed anaerobic reactions for PLA anaerobic biodegradation


Figure 5-2. BMP bottle with anaerobic media


Figure 5-3. Steam-treated ground PLA
















50
-Bm


40

o
u) /
S30



20



10



0
0 50 100 150 200
Dose (kGy)

Figure 5-4. Weight loss of irradiated PLA


- Thermophilic treated PLA
A Thermophilic untreated PLA
- Mesophilic treated PLA
0 Mesophilic untreated PLA
- Theoretical


days

Figure 5-5. Conversion of steam-treated PLA (1200C x 3h) to CH4 under anaerobic conditions









Table 5-1. Production of methane in steam-treated PLA
(1) (2) (3) (4) (5
Time cc gas % CH4 cc CH4 cc
removed removed cu


Syringe
displacement


(2)*(3)/100


C
(4
(5


)

mulative
H4 removed
)t=i +
)t=i-1


(6)
cc CH4 in
headspace

Vhs*(3)/100(a)


Table 5-1. Continued
(8)


(10) (11)


cc CH4 cc CH4 total produced cc CH4 total produced CH4 yield
total @ STP by control by PLA (cc/ g VS)
(7)*(273.15/T)*F(b) Follow steps (1) to (8) (8) (9) (10)/WPLA
for bottles with
control
(a) Estimated in each gas sample analysis
(b) Correction due to pressure vapor: F=(760-pvap)/760. pvap(580C) = 136mm Hg. p,,p(370C) =
47mm Hg [48]

Table 5-2. Weight loss of untreated PLA in water under anoxic conditions
Temperature (OC) Weight loss (%)
37 0.19+0.2
58 98.96+1.5


(7)
cc CH4
total

(5)+(6)









CHAPTER 6
BIODEGRADATION OF STEAM-TREATED PLA UNDER COMPOSTING CONDITIONS

Introduction

Reactions that occur during PLA biodegradation in a composting process occur in three

stages summarized in Figure 6-1. First, PLA hydrolyzes producing lower molecular weight PLA.

This stage requires water and energy, but the presence of microorganisms is not essential. Then,

low molecular weight PLA undergoes production of oligomers and lactic acid. During this

second stage, hydrolysis still occurs but biological activity takes place more intensively, and is

aided by appropriate temperature, and moisture and oxygen levels. The third stage is carried out

only by biological activity and produces carbon dioxide and water [36]. Depending on the pH

and the microbial cultures, radicals could also be produced and combined with the biomass to

integrate humic acids in the compost. Also, a small part of the carbon dioxide fraction transforms

to carbonic acid and bicarbonates transformations due to high moisture levels in the media.

The main objective of this research is to evaluate the effectiveness of different processes as

potential pre-composting treatments for PLA waste, and to determine whether they will allow

complete degradation within the time frame of normal organic compost. In Chapter 2, it was

demonstrated that exposure of PLA to steam is the most effective treatment to reduce molecular

weight and affect structural integrity.

In this chapter, steam-treated PLA samples were subjected to composting conditions to

evaluate their biodegradation, via the method of perforated jars described previously (Chapter 4).

Additional experiments to determine the kinetics of biodegradation were performed.

Results of this work validate the hypothesis of this study, which states that pre-composting

treatments able to reduce PLA molecular weight will be favorable in subsequent composting

processes by reducing overall biodegradation time. An important question is whether









pretreatments provide a "head start" in further composting, and/or contribute to "accelerate"

conversion of PLA.

The "head start" effect is shown in Figure 6-2. A typical curve representing PLA

biodegradation in the composting process can be described through 3 phases: (a) a lag period, (b)

an accelerated biodegradation phase, and (c) a decelerated biodegradation phase until reaching a

plateau. A "head start" effect would shift the curve in time, so that the lag period would be

shortened or eliminated, but the trend of the curve would be maintained. So, a "head start" effect

would be expected to displace the entire curve to the left. As a consequence of this "head start"

effect, overall biodegradation time will be reduced.

The "acceleration" effect is illustrated in Figure 6-3. Here, the biodegradation rate must be

carefully analyzed once the lag period is complete. The slope of the curve (in the earlier phase)

represents initial biodegradation rate, and is an indicator of how rapidly carbon dioxide is

evolving. In Figure 6-3, the dashed curve depicts biodegradation evolution of PLA exhibiting the

"acceleration" effect, represented by a steeper slope.

Material and Methods

Steam-Treated PLA Biodegradation in Compost

Previously (Chapter 4), the method of perforated jars was described. In this section, this

method was used to determine the kinetics of steam-treated PLA biodegradation in compost.

Samples of PLA were ground using an Urschel 3600 grinder (Urschel Laboratories, Inc.,

Valparaiso, IN) with a 3mm screen, and then subjected to steam at 1200C for 0, 1, 2 and 3 hours.

Mason jars of 936cc capacity provided with 5 holes (x 1/16") in the lids were filled with

100g of 6-month mature compost (Appendix A) and 10g of ground PLA samples. Sealed jars

were stored at 580C for 31 days in a Lab-Line L-C Incubator (Lab-Line Instruments, Inc.,

Melrose Park, IL). Beside routine practices such as agitation and moisturizing, concentration of









gases in the headspace was determined daily using a gas analyzer Pac Check 650 (Mocon, Inc.,

Minneapolis, MN). Data collected was processed according to the methodology described earlier

in chapter 4 in order to create curves of PLA biodegradation over time.

Kinetics of Steam-Treated PLA Biodegradation in Compost

Data of biodegradation were plotted and adjusted to the logistic model with three

parameters shown in Equation 6-1. Parameters were estimated using nonlinear regression

performed with SigmaPlot v. 10. Ideally, parameter a should be 100. Parameter b is associated

with the lag period, and the parameter x, represents the time at which half of the biodegradation

would be completed. For untreated PLA, large values of parameters b and Xo were expected,

whereas for treated PLA smaller values were expected.


% biod.= (6-1)
S+(t xo)-b

Weight Loss of Steam-Treated PLA in Compost and Comparison with other Common
Feedstock

Flat sheet samples with rectangular or circular shapes, with similar surface area (-12.5

cm2) were prepared from steam-treated PLA wrapped in nylon screen envelopes, wood and

virgin corrugated paperboard. These samples were dried, weighed and immersed in water for 10

minutes. Wet samples were placed individually into perforated mason jars (cap.936cc)

containing 200g of 6-month mature compost (Appendix A). Controls were jars filled with 200g

compost. Closed jars were stored in a Lab-Line L-C Incubator (Lab-Line Instruments, Inc.,

Melrose Park, IL) for 14 days at 580C.

Periodically, jars were gently shaken to ensure uniform contact of samples with compost,

and water was injected to maintain proper moisture content of the biomass. Samples were

covered by the compost at all times to promote biological activity. At the end of the experiment,









samples were removed from the jars, carefully washed, dried and weighted. Weight loss of each

individual sample was determined using Equation 2-1.

Results and Discussion

Steam-Treated PLA Biodegradation in Compost

Figure 6-4 shows evolution of steam-treated PLA (1200C x 0, 1, 2 and 3 hours)

biodegradation under composting conditions. On average, rates of biodegradation increased as

steam treatment became more severe. These results validate the general hypothesis of this study,

which states that a pre-composting treatment capable of reducing PLA molecular weight would

increase biodegradation rate in subsequent composting process. This figure also confirms the

"head start" and "acceleration" effects, which were postulated as means by which PLA

conversion would be enhanced. As steam treatment increased, "head start" and "acceleration"

effects also increased.

During the experiment, oxygen concentration in the headspace was monitored and found to

be above 17.8% at all times. Agitation permitted good aeration and mixing, but it needed to be

performed carefully in order to minimize clumping of the particles. Unfortunately, clumping

occurred in the jar containing PLA treated with steam for 3 hours at 1200C during the last days

of the experiment. Clumping appeared to slow biodegradation during those last days, probably

due to a lesser area exposed to the environment.

According to standards, "biodegradabililty" requires 60% conversion. In this regard,

samples treated at 1200C for 3, 2 and 1 hours reached biodegradability after 14, 16 and 19 days.

Untreated samples did not achieve biodegradability even after 31 days. Observations of biomass

through glass jars confirmed previous results. Steam-treated PLA at 1200C for 3 hours was no

longer seen in biomass after 14 days of composting. Even when total biodegradation was not yet

achieved, PLA had apparently disappeared, and it could be said that breakdown was complete.









However, continued production of CO2 attributed to PLA material suggests the presence of PLA,

probably as oligomers, and lactic acid.

It was also observed that more severe treatments (i.e. 1200C x 3h) did not create a lag

period for adaptation or conditioning. In these samples, the rate of biodegradation was very high

at the beginning of the experiment and then decreased over time. In contrast, PLA samples less

severely treated (i.e. 1200C x Ih) showed a sigmoidal behavior, represented by a lag period,

accelerating and decelerating stage.

It is said that a material must fulfill three conditions to be compostable [49]: (a) it must

biodegrade quickly, (b) it must not alter the quality of the compost, and (c) it must disintegrate.

Samples of steam-treated PLA fulfilled these conditions. The 31-days biomass, consisting of

biodegraded treated PLA in compost, had similar appearance, texture and odor as compost

without PLA. The pH of the final biomass (-6.6-6.8) did not change (Table 6-1). Pictures of the

compost with and without treated PLA are shown in Figure 6-5. There was no apparent

difference between the two compost samples.

Kinetics of Steam-Treated PLA Biodegradation in Compost

Nonlinear regression to fit experimental data to the logistic model was obtained using

SigmaPlot v.10. Outputs are shown in Table 6-2.

Parameters of the model are related with the pattern and magnitudes of the biodegradation

curves. The parameter a is the plateau, which is the maximum value of biodegradation that can

be achieved. In all cases the value of a is close to 100 which is the theoretical plateau. The

parameter b is associated with the lag period, so smaller values indicate shorter times, and larger

values of b indicate longer times to start biodegrading at a high rate. This matches experimental

results, where more severe pretreatments resulted in lower values of b (for instance, steam-

treated PLA at 1200C x 3h got the shortest value of b, and untreated PLA the highest). Finally,









the parameter x, is the time at which half of the biodegradation will be completed. Thus, larger

values of xo indicate longer total times for biodegradation. This explains why untreated PLA

biodegradation curve had a much higher values of this parameter. The goodness of fit was

excellent in all curves as represented by r-square values very close to unity. Figure 6-6 shows

experimental data and predicted values from the model.

Weight Loss of Steam-Treated PLA in Compost and Comparison with other Common
Feedstock

Figure 6-7 shows results of weight loss of steam-treated PLA (1200C x 3h, 4h), wood and

virgin corrugated paperboard in 6-month mature compost. Treated PLA samples were the only

ones that broke apart inside the compost. Screened envelopes were designed to retain broken

parts for further weighting. After 14 days, steam-treated PLA achieved weight losses of 94.9%

(1200C x 4h) and 86.4% (1200C x 3h), whereas wood and corrugated board achieved values of

0.9% and 39.2%, respectively. These results demonstrate that PLA subjected to steam (1200C x

3 and 4 h) breaks down much faster than wood and virgin corrugated paperboard, which are

usually accepted in composting facilities. Figures 6-8, 6-9 and 6-10 show pictures of these

samples, and it was observed that steam-treated PLA was most greatly affected.

Conclusion

It has been demonstrated that steam-treated PLA is affected very significantly in compost,

breaking down even faster than common organic feedstock universally accepted in composting

facilities such as wood and virgin corrugated paperboard. Polylactic acid treated with steam at

1200C for 3, 2 and 1 hours, achieved degradability (60% of conversion to CO2) after 14, 16 and

19 days, whereas untreated PLA did not achieve biodegradability even after 31 days.

Degradability was evidenced by complete PLA disappearance. Additionally, resulting compost

did not appear to be affected by a loading of about 10% by weight PLA in compost.









Characteristics of the final compost when steam-treated PLA was initially present were similar

than those of the compost by itself. The three requirements for compostability are fast

breakdown, total disintegration and no alteration; thus, steam-treated PLA should be considered

to be compostable.

Biodegradation kinetics of PLA fit very well using the proposed logistic model with three

parameters, and provides valuable information for understanding biodegradation behavior.

Determined parameters confirmed that pre-composting treatments that reduced PLA molecular

weight provided "head start" and "acceleration" effects during subsequent composting process.

Parameter "b" from the model is associated with the lag period and therefore, with the "head

start". The "acceleration" effect is associated with the derivative of the model after the lag

period.


PLA HeM, 0 ris Low MW PLA

Low MW PLA Hea, mo., O oiture P oligomers + lactic acid
Heat, m. O moiitur
Oligomers lactic acid CHeatm CO2 + H20

Figure 6-1. Main reactions in PLA biodegradation









% biod.















Figure 6-2. "Head start" effect


% biod.


Figure 6-3. "Acceleration" effect


Slope 2





























-- Untreated
--120C x 1h
- 120C x 2h
- 120C x 3h


0 5 10 15 20 25 30
Days

Figure 6-4. Biodegradation of steam-treated PLA over time in compost


Figure 6-5. Biodegraded PLA in compost: (a) compost by itself, (b) compost + PLA (not seen
any more)













100





80




o
60

60
"o


.- 40





20


-U


o00
0r0A-2
'0
0 0 Pa



0 /A
A


0 A
9p A' A


o Untreated

S120C x 1h

A 120C x 2h

o120C x 3h


t X_ _


4/


Days

Figure 6-6. Logistic model fit for PLA biodegradation data


PLA 3h @ 120C
Material


Figure 6-7. Weight loss of steam-treated PLA in compost
wood.


compared with corrugated board and


94.9

-86.4









39.2







0.9


PLA 4h @120C


Corr PB


Wood


0 0

























Figure 6-8. Corrugated paperboard subjected to compost for 14 days


Figure 6-9. Wood subjected to compost for 14 days


Figure 6-10. Steam treated PLA (1200C x 3h) subjected to compost for 14 days









Table 6-1. pH of biomass (compost + biodegraded PLA)
Sample pH
Compost 6.7
Oh @ 1200C 6.6
lh @ 1200C 6.6
2h @ 1200C 6.6
3h @ 1200C 6.8


Table 6-2. Parameters of the logistic model (%biod = a / (1 + (t/xo)-b)
Untreated lh @ 1200C 2h @ 1200C 3h @ 120C
a 84.01 75.15 96.91 113.8
b 4.103 3.612 2.04 1.05
Xo 26.45 12.57 12.61 12.49
R-square 0.9819 0.9981 0.9981 0.9981









CHAPTER 7
CONCLUSIONS

Among all the treatments intended to reduce PLA molecular weight, exposure to steam is

the most effective, achieving a reduction of 94% when subjected to steam at 1200C for 4 hours.

The main mechanism for this event is thermal hydrolysis. Gamma irradiation is also a good

treatment for reducing PLA molecular weight, achieving a reduction of 86% when absorbed dose

was 172 kGy.

Steam treatment of PLA is a process that hydrolyzes the polymer molecule until complete

de-polymerization to lactic acid units, as it was confirmed by FTIR-ATR analysis. At 1200C for

24h, PLA weight loss was 84.7% suggesting significant conversion to lactic acid. It is believed

that steam treatments may be suitable not only for making PLA waste more accommodating to

commercial composting operations, but also for assisting with separation of PLA from traditional

plastic wastes. The model that describes molecular weight reduction in PLA as a consequence of

hydrolytic steam treatment follows first order kinetics with activation energy, Ea, of 52.3 KJ/mol.

To evaluate PLA biodegradation in compost, the most reliable and user friendly method

designed and developed was the method of perforated jars. This method does not require a

complex and expensive gas supply system as the one proposed in the standards (ASTM D5338

and ISO 14855) and is much easier to conduct and adapt.

Under anaerobic conditions, untreated PLA did not biodegrade at mesophilic temperature.

Polylactic acid did degrade under thermophilic conditions. Treated PLA, using steam or

irradiation, degraded in mesophilic or thermophilic conditions, and at higher rates than untreated

PLA. For anaerobic digestion, steam-treatment was more effective than gamma irradiation

treatment, and gamma irradiation treatment was more effective than electron-beam irradiation.

The best results observed were for the most severe treatment studied, which was steam at 1200C









for 3 hours, followed by anaerobic digestion under thermophilic conditions, where methane yield

was 225 ccCH4/g after 56 days.

Under aerobic conditions it was confirmed that steam-treatment provided "head start" and

"acceleration" effects to PLA biodegradation. Ground PLA subjected to steam at 1200C for 3

hours achieved biodegradability (60% of conversion as C02) in 14 days, whereas untreated PLA

did not achieve this state even after 31 days. It was verified that steam-treated PLA (1200C x 3h)

undergoes breakdown even faster than virgin corrugated paperboard and wood, common

feedstock universally accepted in composting plants. In addition, at a rate of 10% of compost

feedstock, steam-treated PLA does not appear to alter the compost during the time it is breaking

down.

Three findings of this work are significant. First, it was found that PLA biodegrades under

anaerobic thermophilic conditions. This finding is important because post-consumer PLA

material may be used in anaerobic digestion for energy recovery, instead of being treated as

waste disposal. Second, a simple and reliable method to determine biodegradation of polymers

under composting conditions was developed. This method, named "method of perforated jars,"

may be a valuable contribution to biodegradation assessments and may serve as a more

convenient and less expensive alternative to current standard methods. Finally, steam-exposure

can be also seen as a potential technique for recycling PLA. This is possible since, under steam

conditions, PLA hydrolyzes to lactic acid which is soluble in water, and therefore, may be

separated from the plastic waste stream and re-polymerized into virgin PLA.

There are big differences between biodegradable, bioerodable and compostable. PLA is

slightly biodegradable and bioerodable in composting conditions. However, this is insufficient

for commercial composting operations. Steam treatments may help to separate PLA from









municipal solid waste streams as well as accelerating conversion of the material. Such treatments

may play an important role in helping to keep PLA waste out of sanitary landfills. The ability to

easily compost PLA and/or other biopolymers helps to complete the cycle that is the very

essence of sustainability.









CHAPTER 8
RECOMMENDATIONS FOR FUTURE WORK

Main recommendations for future work are:

Evaluate steam-treated PLA biodegradation in real composting conditions where
temperature varies.

Evaluate lactic acid recovery from a plastic waste downstream containing PLA material,
by using steam treatment.

Find selective microbial strains capable to optimize PLA biodegradation under aerobic,
anaerobic and facultative conditions.

Investigate other ways to recover lactic acid from PLA, such as treating dissolved alkali-
treated PLA.









APPENDIX A
CHARACTERISTICS OF THE COMPOST

The compost was originally developed using a standard organic matter feedstock recipe

developed in this study. It consisted of freshly cut grass from the University of Florida's golf

course (58%), saw dust (11%), virgin corrugated board (11%) and mature compost (20%). All

raw materials were prepared and mixed together in a rotational composer (CompostTwin,

Mantis, Southampton PA) placed in a computer controlled environmental chamber

(Environmental Growth Chambers, Chagrin Falls, Ohio) at 350C .This formulation met the

requirement of initial optimum nutrient balance carbon/nitrogen ratio of 30. Periodic addition of

water provided appropriate moisture content between 55% and 70% to promote and maintain

biological activity. Initial net weight in the composer was 118 lb and rotation was applied daily

to ensure adequate mixing and aeration. Main futures of the 3 months old mature compost are

shown in Table A-1.

Table A-1. Characteristics of prepared mature compost
Features Value Method
Moisture 47-53 % Weight of dry matter/ initial weigh (1050C)
Ash 22-27 % Weight of residue after 5500 for 6 hours
Volatile solids 21-24 % VS = 100 %Moisture %Ash
Size 4.6 mm, max Manual screening
pH 6.8 pH-meter, dilution 1:5
CO2 generation rate 0.14 moles/day/Kg(*) Flow method, variable depending on age

(*) Estimated using the flow method at 3 months old.









APPENDIX B
PERMEABILITY TO CARBON DIOXIDE

To determine the permeability of the plastic film to C02, the cells of the oxygen

transmission rate analyzer Oxtran 2/20 ST (Mocon, Inc., Minneapolis MN) were modified in

order to connect to the carbon dioxide sensor of the headspace analyzer Pac Check 650 (Mocon,

Inc., Minneapolis MN). Carbon dioxide was used in place of oxygen, and the carrier gas was

N2/H2 (96/4). A sketch of the system is shown in Figure B-1.

Permeability to CO2 at 15, 25 and 350C was estimated using Equation B-l after carbon

dioxide concentration, [C02], measured by the sensor, became constant (steady state). The

thickness of the film, E, was measured with a micrometer and was 3.035 mil with an area of 100

cm2. The universal gas constant is R = 82.057 atm-cc/moloK. The gas flow was set at 20cc/min

(at 21.1C, latm) using needle valves of the Oxtran 2/20 ST.

Flowgas x [C02 ]x xE
Pco =(B-l)
RTA


Permeability to CO2 at 580C was estimated using Arrhenius equation shown in Equation

B-2, with Ea = 23.08 KJ/mol and pre-exponential factor Po =58614 mol-mil/atm-d-m2. The final

value obtained was Pco2 @ 580C = 13.41 mol-mil/atm-d-m2.

P = P exp(-E RT) (B-2)


Parameters of the Arrhenius equation, Ea and Po, were determined from linearization of

experimental data of permeability at 15, 25 and 350C. The slope of the plot In Pco2 vs 1/Tis

equal to -E~R, and the intercept is In Po (Figure B-2). The gas constant R for the Arrhenius

equation was R = 0.0083 KJ/K-mol.










Physical cells
(Oxtran 2/20 ST)


C02














CO2 + N2H2
4-


Film


N2H2
4 ---


f--- CO2 sensor
(Pac Check 650)


Flow-meter

N2H2 + C02 I
---______


Figure B-1. Mounted system to determine CO2 transmission rate


1.2 -
0.00320


0.00325 0.00330 0.00335 0.00340 0.00345 0.00350


1/T (K 1)


Figure B-2. Arrhenius plot for activation energy determination (CO2 permeability)









APPENDIX C
VOLUME OF BIOMASS

Volume of biomass inside a container was determined from weight of biomass and its

density (Equation C-1).


V W biomass In container (C-1)
S= v(C-l)
P biomass

To determine density, a biomass sample was inserted and compacted into a vessel of

known weight and volume. Then the filled vessel was weighed and the density was calculated by

the ratio of net weight over volume, as shown in Equation C-2.

W (vessel full) -W(vessel empty)
p = (C-2)
vessel

The vessel used in these analyses was a bottle cap with weight = 5.8728 g and capacity

(volume) = 6.3 cc.









APPENDIX D
BAG VOLUME

Bag volume refers to the internal volume occupied by headspace gases and biomass. Bag

volume, Vbag, was determined by total volume, Vbag tot, minus the volume of the film itself, Vfiim

(Equation D-1).

Vbag Vbag tot Vfim (D-1)

Total volume, Vbag tot, was measured using a volume-meter designed and built for this

study, and uses the principle of water displacement. Figure D-1 describes functionality. Water

warmed to experimental conditions (e.g. 580C) was used as the displacement fluid. Errors due to

depth pressure were predicted to be minimal (< 0.1%).

Volume of the film was calculated by multiplying length by width by thickness of the

opened bag. This was about Vfilm = (12+12)(9)(3.035 x 0.00254) = 1.67 cm3



Radius = r




WI Vagtot T] rrH(R-rl ll .,










Radius = R

Figure D-1. Volume-meter designed for bag volume determination.









APPENDIX E
OVERALL COEFFICIENT OF DIFFUSION

Jars (cap.936cc) with same features as those used for the method of perforated jars

biodegradationn assessment) were filled with 20ml of DI water and some carbon dioxide. Jars

were closed using the 5-hole perforated lids and stored in a Lab-Line L-C Incubator (Lab-Line

Instruments, Inc., Melrose Park, IL) at 580C for 5.5 hours. Each 0.5 h, the headspace was

analyzed for CO2 concentration and the rate of effusion was estimated. Results are shown in

Table E-1.

The percentage of CO2 adjusted was obtained from the empirical exponential model that

best-fitted experimental data. Only those values where %CO2 was below 5.7% were taken into

account for the regression since they better represented values in normal assessments. The

parameters of the model were obtained in MS Excel, with r-square of 0.998 (Figure E-l).

Volume of CO2 adjusted in the headspace expressed in cubic centimeters, is the product of

the CO2 concentration adjusted by the volume of the headspace (936 20 = 916cc). Then, the

number of moles was determined by the Ideal Gases Law (moles = PV/RT) assuming that

expansion of water due to temperature was negligible. Finally, the rate of effusion was calculated

by Equation E-1.

A moles in hs
Effusion rate = (E-1)
Time

Effusion rate was plotted against %C02 in the headspace (Figure 4-12) and the following

relationship was found (Equation E-2);

Effusion rate = 0.00031 (%C02) (E-2)

Equation E-2 shows that the overall coefficient of diffusion is 0.00031 moles C02/h per

percentage unit.










Table E-1. Carbon dioxide effusion rate estimation through 5-hole lid


moles
CO2 in hs
0.01308
0.00617
0.00424
0.00291
0.00200
0.00137
0.00094
0.00065
0.00045
0.00031
0.00021


Effusion rate
moles C02/h
released


Time
(h)
0.0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5


0.0 2.0 4.0 6.0
h
Figure E-1. Carbon dioxide concentration in headspace over time


%C02
exp.
28.1
15.9
11.2
7.8
5.7
4.1
2.9
1.9
1.4
0.9
0.6


%C02
adj.
38.8
18.3
12.6
8.6
5.9
4.1
2.8
1.9
1.3
0.9
0.6


cc CO2
adj.
355.3
167.7
115.2
79.1
54.3
37.3
25.6
17.6
12.1
8.3
5.7


0.00691
0.00386
0.00265
0.00182
0.00125
0.00086
0.00059
0.00041
0.00028
0.00019










APPENDIX F
NUTRIENT FORMULA FOR ANAEROBIC MEDIA


Table F-1. Anaerobic media formulation
Chemical Concentration (g/L)
Resazurin 1
(NH4).2HPO4 26.7
Ca C12.2H20 16.7
H3B03 0.38
H2WO4 0.007
FeCl2.4H20 370
Na2S.9H20 500
Biotin 0.002
Folic acid 0.002
Vitamin B 12 0.0001
Sodium bicarbonate
DI water


Amount
1.80 ml
5.40 ml
27.00 ml
2.70 ml
0.27 ml
18.00 ml
18.00 ml
1.80 ml
0.90 ml
0.18 ml
8.40 g
Up to 2 L









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40. Campanelli J, Kamal M, Cooper D. A kinetic study of the hydrolytic degradation of
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49. De Wilde B, Boelens J. Prerequisites for biodegradable plastic materials for acceptance in
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BIOGRAPHICAL SKETCH

I was born in Lima, Peru, in 1970 where I lived almost all my life. I graduated as Food

Engineer (1992) and MS in Food Technology (1997), both at Universidad Nacional Agraria la

Molina in my home country. In 1995, I married and became a professor at the Food Engineering

Department in my former university. I spent 10 years of my life teaching, training, managing and

researching in the area of food engineering. In 2005, I was awarded a fellowship granted by the

Organization of the American States (OAS) to pursue the Ph.D. program at University of Florida

through the Agricultural and Biological Engineering Department / Packaging Science Program. I

have spent my last three years studying topics related to packaging and doing research in the

field of biodegradable packaging, under Dr. Bruce Welt.





PAGE 1

EFFECT OF PRE-TREATMENTS ON THE KI NETICS OF SUBSEQUENT AEROBIC AND ANAEROBIC BIODEGRADATION OF POLYLACTIC ACID (PLA) By LUIS FERNANDO VARGAS DELGADO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Luis Fernando Vargas Delgado 2

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To my wife, son and parents 3

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ACKNOWLEDGMENTS I express my sincere appreciation to my a dvisor Dr. Bruce Welt. Definitely, he is an exceptional professor and a good friend, very dedicat ed and always happy to support me with his valuable guidance. I am gratef ul to my committee members Dr. Art Teixeira, Dr. Balaban, Dr. Pullammanappallil, and Dr. Beatty for their orientation. I thank my colleagues Richlet Dorcent, Ayman Abdellatief and Cecilia Amador for their collaboration and friendship. I express my gratitude to Steve Feagle, James Rummell and B illy Duckworth whose technical expertise was very valuable for this research. Also, I was happy to interact with my colleagues from the bioprocess engineering lab, who were always av ailable to assist me. I show my sincere appreciation to the University of Florida and the Agricultur al and Biological Engineering Department for their funding and support. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.........................................................................................................................9 LIST OF ABBREVIATIONS........................................................................................................12 ABSTRACT...................................................................................................................................14 CHAPTER 1 INTRODUCTION................................................................................................................ .....16 2 EVALUATION OF TREATMENTS TO REDUCE PLA MOLECULAR WEIGHT.............18 Introduction................................................................................................................... ..........18 Materials and Methods...........................................................................................................19 Materials..........................................................................................................................19 Methods of Analysis........................................................................................................19 Subjective assessment of structural integrity...........................................................19 Determination of weight loss...................................................................................19 Determination of molecular weight..........................................................................20 Determination of degree of chain scission...............................................................20 Determination of mechanical properties..................................................................21 Exposure of PLA to Gamma Irradiation.........................................................................21 Exposure of PLA to Electron Beam Irradiation..............................................................21 Immersion of PLA in Alkaline Media.............................................................................23 Immersion of PLA in Acid Media...................................................................................24 Exposure of PLA to Steam..............................................................................................24 Results and Discussion......................................................................................................... ..24 Exposure of PLA to Gamma Irradiation.........................................................................24 Mechanical properties..............................................................................................24 Molecular weight......................................................................................................25 Degree of chain scission...........................................................................................26 Exposure of PLA to Electron Beam Irradiation..............................................................26 Before composting...................................................................................................26 Structural integrity....................................................................................................26 Molecular weight......................................................................................................27 Degree of chain scission...........................................................................................27 After composting......................................................................................................28 Structural integrity....................................................................................................28 Weight loss...............................................................................................................29 Molecular weight......................................................................................................30 5

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D-value.....................................................................................................................30 Immersion of PLA in Alkaline Media.............................................................................31 Structural integrity....................................................................................................31 Weight loss...............................................................................................................31 Molecular weight......................................................................................................32 Immersion of PLA in Acid Media...................................................................................32 Structural integrity....................................................................................................32 Weight loss...............................................................................................................32 Exposure of PLA to Steam..............................................................................................32 Structural integrity....................................................................................................32 Molecular weight......................................................................................................33 Conclusions.............................................................................................................................34 3 KINETICS OF REDUCTION OF MO LECULAR WEIGHT IN STEAM-TREATED PLA.........................................................................................................................................47 Introduction................................................................................................................... ..........47 Methods..................................................................................................................................47 First Order Reaction Model.............................................................................................47 Depolymerization of PLA to Lactic Ac id Resulting from Steam Exposure...................48 Results and Discussion......................................................................................................... ..49 First-Order Reaction Model............................................................................................49 Depolymeryzation of PLA to Lactic Ac id Resulting from Steam Exposure..................49 Conclusion..............................................................................................................................50 4 DEVELOPMENT OF METHODS TO EVALUATE AEROBIC BIODEGRADATION OF PLA...................................................................................................................................54 Introduction................................................................................................................... ..........54 Materials and Methods...........................................................................................................55 Method of Flexible Bags.................................................................................................55 Method of Rigid Containers with Plastic Film Lids........................................................57 Method of Perforated Jars...............................................................................................58 Results and Discussions........................................................................................................ ..59 Method of Flexible Bags.................................................................................................59 Method of Rigid Containers with Plastic Film Lids........................................................61 Method of Perforated Jars...............................................................................................61 Conclusion..............................................................................................................................62 5 BIODEGRADATION OF TREATED PL A UNDER ANAEROBIC CONDITIONS.............71 Introduction................................................................................................................... ..........71 Materials and Methods...........................................................................................................72 Weight loss of PLA in Water under Anoxic Conditions.................................................72 Weight Loss of Irradiated PLA in Anaerobic Biological Media.....................................72 Biochemical Methane Potential (BMP) of Steam-Treated PLA in Anaerobic Biological Media..........................................................................................................73 6

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Results and Discussion......................................................................................................... ..74 Weight Loss of PLA in Water Under Anoxic Conditions...............................................74 Weight Loss of Irradiated PLA in Anaerobic Biological Media.....................................74 Biochemical Methane Potential (BMP) of Steam-Treated PLA in Anaerobic Biological Media..........................................................................................................75 Conclusions.............................................................................................................................76 6 BIODEGRADATION OF STEAMTREATED PLA UNDER COMPOSTING CONDITIONS..................................................................................................................... ...80 Introduction................................................................................................................... ..........80 Material and Methods.............................................................................................................81 Steam-Treated PLA Biodegradation in Compost............................................................81 Kinetics of Steam-Treated PLA Biodegradation in Compost.........................................82 Weight Loss of Steam-Treated PLA in Compost and Comparison with other Common Feedstock.....................................................................................................82 Results and Discussion......................................................................................................... ..83 Steam-Treated PLA Biodegradation in Compost............................................................83 Kinetics of Steam-Treated PLA Biodegradation in Compost.........................................84 Weight Loss of Steam-Treated PLA in Compost and Comparison with other Common Feedstock.....................................................................................................85 Conclusion..............................................................................................................................85 7 CONCLUSIONS........................................................................................................................92 8 RECOMMENDATIONS FOR FUTURE WORK....................................................................95 APPENDIX A CHARACTERISTICS OF THE COMPOST...........................................................................96 B PERMEABILITY TO CARBON DIOXIDE............................................................................97 C VOLUME OF BIOMASS.........................................................................................................99 D BAG VOLUME......................................................................................................................100 E OVERALL COEFFICI ENT OF DIFFUSION........................................................................101 F NUTRIENT FORMULA FOR ANAEROBIC MEDIA..........................................................103 LIST OF REFERENCES.............................................................................................................104 BIOGRAPHICAL SKETCH.......................................................................................................108 7

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LIST OF TABLES Table page 2-1 Linear regression outputs and D-values of e-beam irradiated/composted PLA................46 2-2 Comparison of treatments to reduce PLA molecular weight.............................................46 4-1 Biodegradation of PLA by method of flexible bags..........................................................69 4-2 Biodegradation of PLA by th e method of perforated jars..................................................69 5-1 Production of methane in steam-treated PLA....................................................................79 5-2 Weight loss of untreated PLA in water under anoxic conditions......................................79 6-1 pH of biomass (compost + biodegraded PLA)..................................................................91 6-2 Parameters of the logistic model (%biod = a / (1 + (t/xo)-b)..............................................91 A-1 Characteristics of prepared mature compost......................................................................96 E-1 Carbon dioxide effusion rate estimation through 5-hole lid............................................102 F-1 Anaerobic media formulation..........................................................................................103 8

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LIST OF FIGURES Figure page 2-1 Drinking cups made of PLA..............................................................................................35 2-2 Principle of intrinsic viscosity met hod for determination of molecular weight................35 2-3 Stress at break of -irradiated PLA samples......................................................................36 2-4 Strain at break of -irradiated PLA samples......................................................................36 2-5 Molecular weight of -irradiated PLA samples.................................................................37 2-6 Determination of Gs in PLA -irradiation..........................................................................37 2-7 E-beam irradiated PLA showing voids formation.............................................................38 2-8 Molecular weight of e-beam irradiated PLA.....................................................................38 2-9 Determination of Gs in PLA e-beam irradiation................................................................39 2-10 Temperature profile of compost bulk................................................................................39 2-11 E-beam irradiated PLA after 6 weeks in compost.............................................................40 2-12 Structural integrity of ebeam irradiated/composted PLA.................................................40 2-13 Weight loss of e-beam irradiated/composted PLA............................................................41 2-14 Molecular weight of e-beam irradiated/composted PLA...................................................41 2-15 Hydrolysis of PLA......................................................................................................... ....42 2-16 Plots of e-beam irradiation dose vs. log Mw for D-values estimation................................42 2-17 Structural integrity of PL A subjected to alkaline media....................................................43 2-18 Weight loss of PLA subjected to 0.1N NaOH...................................................................43 2-19 Molecular weight of PLA subjected to alkaline media......................................................44 2-20 Structural integrity of steam-treated PLA..........................................................................44 2-21 Pores in steam-treated PLA...............................................................................................4 5 2-22 Molecular weight of steam-treated PLA............................................................................45 3-1 First-order reaction plots for steam-treated PLA...............................................................51 9

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3-2 Arrhenius plot for steam-treated PLA................................................................................51 3-3 Spectra of lactic aci d obtained by FTIR-ATR...................................................................52 3-4 Mass balance of PLA treated w ith steam at extreme conditions.......................................52 4-1 Set up for plastic biodegradation assessment in compost ASTM D5538........................63 4-2 Interactions in flexible bags.............................................................................................. .63 4-3 Pictures of flexible bags with biomass biodegrading........................................................64 4-4 Chemical formula of PLA..................................................................................................64 4-5 Interactions in rigid cont ainer with plastic film lid............................................................65 4-6 Picture of rigid contai ner with plastic film lid...................................................................65 4-7 Interaction in perforated jar.............................................................................................. .66 4-8 Picture of jar filled with biomass and perforated lid..........................................................66 4-9 Picture of compost plus PLA afte r 40 days, in and out of the bag....................................67 4-10 Biodegradation of PLA using the method of flexible bags................................................67 4-11 Biodegradation of PLA using the method of rigid containers with plastic film lids.........68 4-12 Overall effective coe fficient of diffusion for CO2 through 5 holes...................................68 4-13 Biodegradation of PLA using the method of perforated jars.............................................69 5-1 Supposed anaerobic reactions for PLA anaerobic biodegradation....................................77 5-2 BMP bottle with anaerobic media......................................................................................77 5-3 Steam-treated ground PLA................................................................................................77 5-4 Weight loss of irradiated PLA...........................................................................................78 5-5 Conversion of steam-treated PLA (120C x 3h) to CH4 under anaerobic conditions.......78 6-1 Main reactions in PLA biodegradation..............................................................................86 6-2 Head start effect..............................................................................................................87 6-3 Acceleration effect...................................................................................................... ...87 6-4 Biodegradation of steam-treated PLA over time in compost.............................................88 10

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6-5 Biodegraded PLA in compost............................................................................................88 6-6 Logistic model fit for PLA biodegradation data................................................................89 6-7 Weight loss of steam-treated PLA in co mpost compared with corrugated board and wood...................................................................................................................................89 6-8 Corrugated paperboard subj ected to compost for 14 days.................................................90 6-9 Wood subjected to compost for 14 days............................................................................90 6-10 Steam treated PLA (120C x 3h) subjected to compost for 14 days.................................90 B-1 Mounted system to determine CO2 transmission rate........................................................98 B-2 Arrhenius plot for activation energy determination (CO2 permeability)...........................98 D-1 Volume-meter designed fo r bag volume determination..................................................100 E-1 Carbon dioxide concentration in headspace over time....................................................102 11

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LIST OF ABBREVIATIONS A : Area (cm2) D : Irradiation dose (kGy) Def : Coefficient of diffusion (mol CO2/h/%) E : Thickness of film (mil) Ea : Activation energy (KJ/mol) Gs : Degree of chain scission (w/o units) k : Reaction rate constant or kinetic constant (h-1) ko : Pre-exponential factor (h-1) M : Molecular weight (g/mol) Mn : Number-average molecular weight (g/mol) Mn, o : Number-average molecular weight, initial (g/mol) Mv : Viscosity-average molecular weight (g/mol) Mw : Weight-average molecular weight (g/mol) NA : Avogadro number = 1.023 x 1023 P : Permeability (mol-mil/atm-day-m2) PCO2 : Permeability to CO2 (mol-mil/atm-day-m2) Po : Pre-exponential factor for permeability (mol-mil/atm-day-m2) patm : Pressure, atmospheric (atm) pCO2 : Partial pressure of CO2 (atm) pvap : Vapor pressure (atm) R : Gas Law constant (82.057 at m-cc/molK or 8.31 J/molK) T : Temperature, absolute (K) t : Time (h or days) 12

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V : Volume (cc) Vhs : Volume of headspace (cc) Vbag : Volume of bag (cc) Vbag tot : Volume of bag, total (cc) Vfilm : Volume of film (cc) W : Weight (g) Wo : Weight, initial (g) Wf : Weight, final (g) w : Weight loss (%) : Density (g/cc) : Number of moles red : Reduced viscosity (ml/g) [ ] : Intrinsic viscosity (ml/g) % CO2 : Percentage of CO2 (%) [CO2] : Concentration of CO2 (cc CO2/ cc total) 13

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECT OF PRE-TREATMENTS ON THE KI NETICS OF SUBSEQUENT AEROBIC AND ANAEROBIC BIODEGRADATION OF POLYLACTIC ACID (PLA) By Luis Fernando Vargas Delgado May, 2008 Chair: Bruce Welt Major: Agricultural and Biological Engineering The purpose of this work was to evaluate tr eatments that increase polylactic acid (PLA) biodegradation during composting and anaerobic digestion. Treat ments were chosen on the potential to reduce molecular weight and affect st ructural integrity of the polymer. Five potential treatments were investigate, and it was found that steam exposure was the most effective treatment with up to 94% molecular weight reduction with 120C steam for 4 hours. To determine kinetics of PLA molecular weig ht reduction after exposur e to steam, a firstorder reaction model was proposed. It was found that the model, which is time and temperature dependent, fits experimental data well, with activ ation energy, Ea, of 52.3 KJ/mol. In addition, it was demonstrated that steam treatments hydrolyze polymer molecules, resulting in depolymerization to lactic acid. At 120C, weight loss of PLA wa s 84.7% after 24 hours, indicating significant conversion to lactic acid. To develop a simple method to evaluate PLA aerobic biodegradation in compost, the fundamentals of conversion were examined, and three approaches were designed and assessed. These methods are referred to as methods of flexi ble bags, rigid contai ners with plastic lids 14

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and perforated jars. It was found that the method of perforated jars was most reliable, simple and consistent to apply. To evaluate effects of treatment in subs equent anaerobic digestion, weight loss and biochemical methane potential (B MP) of treated samples were investigated under mesophilic and thermophilic anaerobic conditions. Untreated PL A did not degrade under mesophilic conditions. However, degradation did occur under thermophilic conditions, producing 187 ccCH4/g at 58C after 56 days. The best scenario evaluated, was steam-treated PLA at 120C for 3 hours subjected to anaerobic thermophilic conditi ons, where the yield of methane was 225 ccCH4/g after 56 days. While untreated PLA biodegrades more slow ly than common orga nic feedstock, steamtreated PLA biodegraded faster. Re sults of this investigation show that treatment of PLA with steam at 120C for 3 hours reached 60% degrad ation in 14 days. Compost was not altered by PLA conversion. 15

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CHAPTER 1 INTRODUCTION Recent efforts to improve sustainability imply, among other things, using renewable resources, reducing municipal waste and avoidi ng solid, liquid (incl uding oils) and gaseous emissions [1]. Plastic packagi ng, while providing valuable cont ributions to society is often perceived as a solid waste burden. Traditional plastic packaging does contribute to virtually nondegradable solid waste in la ndfills. In addition, most polymers are petroleum-based, and therefore, obtained from non-renewable resource s. These issues have sparked interest in biobased and biodegradable plastics [2]. Polylactic acid (PLA) is a biodegradable plastic polymer derived from renewable resources, such as corn, that can be used in a variety of packaging applications. During industrial composting, PLA is assu med to degrade through biological activity to carbon dioxide and water. Compostability of PLA has been discussed by different authors, and the agreement is that it truly occurs at te mperatures around 58C after se veral weeks [3, 4, 5]. These characteristics coupled with rece nt capital investments to produ ce commercial quantities of the material have made PLA a viable option for re placing fossil fuel derived polymers for certain applications [6]. As increasing amounts of PLA are finding pack aging applications, a number of practical challenges have become apparent including PLAs instability at moderate temperature and relative humidity [7, 8], and PLAs relatively low biodegradation rate relative to typically composted organic wastes, which is preventi ng commercial compost operators from accepting PLA. Therefore, most post-consumer PLA is being sent to landfills for disposal where breakdown does not readily occur [9]. Since comp anies promote PLAs sustainable qualities, consumers expect that PLA waste will be either composted or recycled, thus completing the 16

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sustainable cycle. Sending PLA to sanitary la ndfills threatens to violate consumer and public trust in sustainable packaging materials. Biodegradation is a two-step process. First, polymer molecules are hydrolyzed into smaller pieces resulting in lower molecular weight polymers, oligomers and monomers. Second, biological action of microorganism s metabolizes these smaller mol ecules resulting in conversion to carbon dioxide and water [10]. Therefore, it is suspected that treatme nts capable of rapidly reducing PLA molecular weight will allow biological conversion s ooner, which should lead to an overall reduction in biodegradation time. The motivating hypothesis of this work is that treatments capable of disrupting the polymer matrix and/or reducing molecular weight s hould result in reduced overall composting time. Additionally, in the event that composting time is reduced, experiments were designed to determine whether pretreatments accelerated conv ersion kinetics or simply provided a head start to the composting process that subsequently pr oceeded at the typical rate. Treated PLA will already be constituted by smaller molecules when added into the composter, so less time is needed to get to this point. The acceleration c oncept is based on increased metabolic activity associated at higher concentrations of lower molecular weight polymer. The main objective of this research was to evaluate effects of pot ential pretreatments on kinetics of subsequent PLA aerobic and anaerobic biodegradation, and to determine whether or not treatments will allow PLA to completely degrade within the time frame of normal organic feedstock. Secondary objectives were to develop a methodology to measure PLA biodegradation rate in a composting process and to determine kinetics of molecu lar weight reduction in treated PLA. 17

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CHAPTER 2 EVALUATION OF TREATMENTS TO REDUCE PLA MOLECULAR WEIGHT Introduction Composting is a well known process that is mainly used to break down organic materials such as yard and food wastes. While PLA is su sceptible to breakdown during composting, it has been found that PLA degradation kinetics are considerably slower than typical organic feedstocks, and currently, little, if any PLA waste is sent to industrial compos ting facilities. Commercial PLA packages have been shown to be incompletely degraded after 28 days of composting [3, 4] and recycling is currently not taking place [5]. Therefore, PLA represents a potential bottleneck to compos ting operations, which could resu lt in PLA being diverted to sanitary landfills for disposal. In this regar d, PLAs promise of sustainability would not be completely fulfilled. Therefore, a key question for compostable sustainable plastics is how to improve degradation kinetics without compromisi ng important useful qualities of the polymer. Some authors have reported on effects of gamm a irradiation [11], elec tron beam irradiation [12], enzymatic hydrolysi s [13] and chemical hydrolysis on pol y (L-lactide) acid (PLLA) [14]. Results showed reductions in molecular weight a nd associated loss of tensile strength, which are indicators of polymer degradation. It is believed that such initial damage to the polymer will help to at least provide a head start and may actua lly accelerate degradation kinetics of PLA during composting. This chapter evaluates potential treatments capable of reducing PLA molecular weight. For this purpose, five treatments were chosen: (1) exposure to gamma irradiation, (2) exposure to electron beam irradiation, (3) immersion in alkali ne media, (4) immersion in acid media and (5) exposure to saturated steam. Additionally, other anal yses such as structural integrity and weight 18

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loss were also performed. These treatments were compared and the most effective was selected for further investigation under comp osting conditions (Chapter 6). Materials and Methods Materials Thermoformed PLA drinking cups (Fabri-Kal Inc., Kalamazoo, MI) were obtained from TREEO Center at the University of Florida (Fig ure 2-1). Cup dimensions were measured using a caliper (Mitutoyo Model CD-6 CS, Mitutoyo Corp., Japan) and are provided in Figure 2-1. Wall thicknesses were 150-200m and bottoms were about 750m. Methods of Analysis Subjective assessment of structural integrity Polymer appearance and physical characteristi cs changed significantly due to various treatments. Descriptive observation was used to compare samples at specific times during each study. Assessment consisted of evaluation by touch, si ght, physical attributes such as brittleness, opacity, whitening, voids/pores formation, swelli ng, twisting, curling, conversion to powder and weakening. A digital microscope (INTEL m odel APB-24221-99A, Mattel Inc., China) at magnifications of 10X, 60X and 200X was used to enhance observational power. Determination of weight loss Weight loss ( w ) was calculated as a per centage using Equation 2-1. %100 W WW wo o (2-1) A precision scale (Voyager Pro/Ohaus, Oh aus Corp., Pine Brook, NJ) was used for individual weight measurements. In all cases at least three repetitions we re performed, but since there was too much variability, only averages are presented in the graph. PLA samples were carefully selected, cleaned and dried. 19

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Determination of molecular weight Intrinsic viscosity was used to determine weight-average molecular weight, Mw, and viscosity-average molecular weight, Mv, of PLA samples in accordance to ASTM D2857-95 [15]. Kinematic viscosities, of PLA dilutions (up to 1% V/V )) were determined at 30C using chloroform as solvent and a calibrated capillary viscosimet er (Cannon-Ubbelodhe Type N, State College, PA). These values were used to determine reduced viscosity, red, and intrinsic viscosity, [ ] of each treated sample. Finally, molecular weight, M was estimated using the Mark-Houwink model (Equation 2-2), which relates molecular weight to the intrinsic viscosity. Figure 2-2 shows the principle of this method. akM][ (2-2) Constants, k and a, used for PLA in chloroform at 30C were 0.0131ml/g and 0.777 for Mv, and, 0.0153ml/g and 0.759 for Mw [16]. At least two repetitions were performed for analysis of gamma and e-beam irradiated (not composted) PLA. Determination of degree of chain scission It is well known that irradiation of polymers causes two main and opposing effects including, scission and crosslinki ng. The important physical fact is that one chain scission causes one molecule to become two, and one crosslink causes two molecules to become one [17]. To evaluate the predominance of chai n scission rather than crosslinking, the concept of degree of chain scission, Gs, was applied. This is defined as the num ber of radiolysis events caused by the absorption of 100eV of radiation and can be ca lculated by Equation 2-3 [11]. Number-average molecular weight, Mn, was assumed to be equal to viscosity-average molecular weight, Mv, since doses were below the gel dose [18]. High values of Gs indicate chain scission predominance over crosslinkings. 20

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D1024.6 ) M 1 M 1 (N Gs16 o,nn A (2-3) Determination of mechanical properties A 4301-Series Instron was used to determine stre ss and strain at break at a temperature of 25C in PLA samples. The equipment was set at a crosshead speed of 30 mm/min, and samples set in the machine direction with a gage lengt h of 30 mm. At least fi ve repetitions were performed in each tensile test to obtain repr esentative average values. This analysis was conducted for PLA samples exposed to gamma irradiation. Exposure of PLA to Gamma Irradiation Rectangular thin sheets (50mm x 10mm x 150-200 m) and shredded pieces (~4mm edge) obtained from PLA cup walls (F igure 2-1) were prepared and irradiated in a foil lined paperboard canister (73 mm diam eter 180 mm height). The can ister was placed inside the irradiation chamber (FL Accelerator Services and Technology, Gainesville, FL) where it was exposed to -rays from a Cesium-137 source at a dose rate of 0.78 kGy/h. Samples were irradiated in air in order to promote scission over crossli nking [11, 17]. The canister was removed from the irradiation chamber after 92 and 221 hours. Dosimetry showed that samples achieved absorbed doses of 72 kGy and 172 kGy, re spectively. Irradiated samples were kept in storage at 25C for at least 4 days prior to anal ysis. Mechanical propertie s (stress and strain at break), molecular weight and degree of ch ain scission were subsequently assessed. Exposure of PLA to Electron Beam Irradiation Electron beam irradiation was applied to PLA samples in order to evaluate effects before and after composting. Square sheets (31.5mm x 31.5 mm x 0.75mm) obtaine d from flat bottoms of PLA drinking cups (Figure 2-1) were prepared and subjected to electron beam irradiation (FL 21

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Accelerator Services and Technology, Gainesville, FL) at a dose rate of 2.4 kGy/min. Bunches of PLA samples were placed together in Petri dishes that resulted in less than 1 cm overall thickness [19]. Samples were transported by conveyor into the irradiation chamber for each irradiation pass. Multiple passes were requir ed to achieve desired doses. Since the goal was to induce chain scissions, and since scission is aided by oxygen [11] no effort was made to protect samples from oxygen. Samples were removed from the irradiati on tunnel after 2, 4 and 6 passes at 36kGy per pass, achieving absorbed doses of 72, 144 and 216 kG y. Irradiated samples were kept in storage at 25C at least 4 days prior to analysis. Elec tron beam irradiated samples were evaluated for structural integrity, molecular wei ght and degree of chain scission. After e-beam irradiation, PLA samples were subjected to composti ng. For this purpose, individual, previously e-beam irradiated samples, were placed inside hand-made, heat sealed nylon screen envelopes and placed in a ro tational composter (C ompostTwin, Mantis, Southampton, PA) equipped with thermocouples in a computer controlled environmental chamber (Environmental Growth Chambers, Chagri n Falls, OH) at 35C. A standard organic feedstock recipe was developed for this study an d used throughout. Organic feedstock consisted of freshly cut grass from the University of Floridas golf course (58%, C:N=10), saw dust (11%, C:N=500), virgin corrugated paperboard (11% C:N=170) and mature compost (20%). Raw materials were sliced or cut in order to achieve a homogeneous mixture. Also, this formulation met the requirement of initial optimum nutrien t balance carbon/nitrogen ratio of 30. Water was periodically added to the mixtur e in order to maintain moistu re content between 55% and 70%, and to promote and maintain biological activity [20]. Initial net weight in the composter was about 118 lb and was rotated daily to ensure adequate mixing and aeration. 22

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Screened sample envelopes allowed for intimate contact between samples and bulk compost and protected against loss of non-mine ralized sample during critical phases of decomposition. Thermocouples inside the composter measured temperature of the composting biomass over 10 weeks. Structural integrity, weight loss, mol ecular weight and irradiation induced molecular weight loss D-value were determined in PLA samples for assessment of electron beam irradiation effect on them. Irradiation D-values are often used to de scribe microbial inactivation. A D-value represents the dose required to cause one log cycle change in a measured parameter [21, 22, 23], which means reducing its value to its 10%. Here, we used D-value to describe the irradiation dose required to change molecular weight by one log cycle. D-values were calculated for composted samples after 0, 1, 2 and 6 weeks. D-valu es were determined from inverses of slopes from linear regressions of log10 Mw versus absorbed dose. Therefore, steeper curves represent lower D-values and greater sensitivity of molecular weight to dose. Immersion of PLA in Alkaline Media Rectangular sheets (6cm x 4cm x 0.75mm) obtained from PLA drinking cup bottoms (Figure 2-1) were placed in 50 ml 0.1N NaOH and 50 ml of 1N Na OH, and stored for 22 days at 25C. Samples immersed in 0.1N NaOH were evalua ted over time for structural integrity, weight loss, and weight-average molecular weight. In itially, the experimental design considered evaluating both conditions (0.1N and 1N), however, PLA samples immersed in 1N NaOH fragmented in such small parts that it was impossible to monitor weight loss or obtain a representative sample for molecu lar weight analysis. Therefore, the only samples evaluated were those subjected to 0.1N NaOH. 23

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Immersion of PLA in Acid Media Rectangular sheets (6cm x 4cm x 0.75mm) obtained from PLA drinking cup bottoms (Figure 2-1) were placed in 100m l of aqueous solution of nitric acid at 0.1% or 0.016N (pH 1), and stored for 22 days at 25C. At the beginning and end of the experiment structural integrity and weight loss were assessed. Change in molecula r weight was not evaluated because results of previous analyses proved that PLA sa mples were not sensitive to acids. Exposure of PLA to Steam Rectangular sheets (6cm x 4cm x 0.75mm) obt ained from PLA cup bottoms (Figure 2-1) were prepared and placed inside jars. Lids were adapted with two holes (about 1cm diameter) to allow steam transfer. Jars were placed in a vertical still retort where steam was fed and temperature/pressure was controlled with a pneum atic system. Experiments were run at 100, 110 and 120C, for 1, 2, 3, 4 and 8 hours. After each treatment, samples were quickly cooled in air to room temperature and dried in an oven at 105C until constant weight. St ructural integrity and molecular weight of steam-treat ed PLA samples were evaluated. Results and Discussion Exposure of PLA to Gamma Irradiation Mechanical properties PLA made from pure L-Lactide, also called poly(L-lactide), is semi-crystalline. Incorporation or co-polymerization with isomer s M-lactide or D-lactid e decreases degree of crystallinity, causing polymers to become more amorphous [24, 25] PLA resins can be tailormade for different fabrication processes, incl uding injection molding, sheet extrusion, blow molding, thermoforming, film forming, or fiber spinning. Critical factor s include degree of branching, D-isomer content, and molecular we ight distribution. For thermoforming, D-isomer 24

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content might be in the range of 4% [25]. Uni rradiated PLA mechanical properties have been studied by others and comparisons to oriented polystyrene (OPS) have been made [26]. Rectangular PLA samples showed increasing brittleness with absorb ed dose. At higher doses, samples were sensitive to even careful handling. Results for stress and strain at break (rupture strength) using the Inst ron 4301 are shown in Figure 2-3 and Figure 2-4. Higher gamma irradiation doses reduced PLA mechanical properties in a manner that is similar to traditional thermoplastic polymers including polystyrene, polypropylene and polyurethane [27, 28, 29]. Other authors studied effects of electron b eam irradiation on lactides and found similar trends [12, 30]. Stress at break of PLA samples irradiated at 172 kGy dropped from 127 MPa to 18MPa, a factor of about 7. For the same sample s, strain at break dropped from 75% to 2%. These numerical values represent a marked increase in brittleness of the irradiated PLA samples. This behavior suggests crystalline dama ge due to free radical attack [12]. Molecular weight Figure 2-5 shows weight-average molecular weight, Mw, and viscosity-average molecular weight, Mv, for un-irradiated and -irradiated PLA samples. Since intrinsic viscosities are lower at higher absorbed doses, molecular weights al so conform to the Mark-Houwink equation. This result confirms that chain scission was the predom inant effect of tested irradiation treatments. Weight-average molecular weight dropped 86% from 16.34 to 2.34. Number-average molecular weight dropped 85% from 15.14 to 2.24. These molecular weight reductions were more severe than those found by others fo r poly(L-lactic) acid [11]. In this work, the material was commercial thermoformed PLA that includes D-isomers, and this may be the reason for such difference. 25

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Ratios of weight average to visc osity average molecular weight, Mw/ Mv, were 1.08, 1.05 and 1.03 for absorbed doses of 0, 72 and 172 kGy, respectively. These narrow molecular weight distributions tend to suggest that PLA used fo r these samples was created from a ring-opening polymerization process. This happens to be th e process claimed by the PLA manufacturer [25]. Degree of chain scission The plot of 1/Mn 1/Mn,0 vs. dose is shown in Figure 2-6. The straight lin e suggests that chain scission was random [31]. Chain scission yield, Gs, was found to be 2.21, which is greater than values found by other researchers for gamma irradiated poly (L-lactic ) acid [11], e-beam irradiated poly (lactide-co-glycolide) and e-b eam irradiated poly (L-lactide) [11, 12]. This suggests that gamma irradiation has a higher effect on chain scission than e-beam irradiation, and that inclusion of D-isomers in the poly mer structure increase such sensitivity. Exposure of PLA to Electron Beam Irradiation Before composting Structural integrity Figure 2-7 shows digital pictures at magnifications of 10X of irradiated PLA surface. Mainly, four phenomena occurred after e-beam exposure: (a ) twisting, (b) change of color, (c) voids formation and, (d) structural weaknesses. Also, it was observed that these changes were more severe as irradiation doses increased. Twisting may be explained by the relatively high irradiation dose rate resulting in temperature increase and associated relaxa tion of polymer chains. The glass transition temperature for PLA has been reported to be around 58C [10]. Sample color changed from transparent to yellow, however, transparency re turned after about one week. Irradiated polymers form unstable chromatic groups, possibly by th e introduction of conjugation to the carbonyl groups of the chain [32]. It is well known that radiation induced ch emistry continues after 26

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exposure and this is a reason that plastic radiation dosimeters require at least 48 hours before reaching final significant changes. The phenomenon related with voids formation is common in abused irradiated materials. Generally, there should be void nuc lei formation followed by swelling, which is attributed to an increase of temperature and irradiation dose [33]. Irradiated PLA samples showed internal swelling voids that increased in number and size as irradiation dos e increased. Void formation is likely the result of a combination of chain sc ission, gas formation, swelling, and associated polymer rearrangement due to heating. Polylactic acid samples became extremely br ittle, suggesting a reduction in chain length with irradiation dose. This behavior also sugge sts damage to crystallin e regions due to free radical attack [12]. Molecular weight Figure 2-8 shows how PLA molecular weight was affected by e-beam irradiation. PLA samples treated with 0, 72, 144 and 216 kGy of e-beam irradiation achieved weight-average molecular weights of 1.7x105, 8.2x104, 6.9x104 and 6.1x104 g/mol. These values represent about 48%, 40% and 35% of the ini tial molecular weights. Comparing with results for gamma irradiation (Mw at 172 kGy = 14% of untreated Mw), it can be said that PLA is more vulnerable to ga mma rather than e-beam irradiation. For any of these treatments, decreases in molecular weight o ccur due to scission of the main backbone [12]. Degree of chain scission, Gs The plot of 1/Mn 1/Mn,0 vs. dose, D is shown in Figure 2-9. Th e degree of chain scission, Gs, was found to be 0.52, which is lower than the 2.21 value obtained for gamma irradiation using Cs-137. It is likely th at higher dose rates involved w ith electron beam irradiation 27

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effectively reduced local oxygen concentrations, wh ich helped to protect polymer samples during irradiation. After composting After 2 weeks of composting, most organic feedstock biomass excluding PLA looked and smelled as finished compost. Literature indicat es that this aerobic bioprocess releases water vapor (0.6-0.8 g/g), carbon dioxide and heat (about 25 KJ/g), which cause the compost temperature to rise significantly [20]. Figure 2-10 shows that co mpost temperatures quickly rose to about 68C during the first week, fell to 45C by the second week, and leveled off at the chamber controlled temperature of 35C by the third week. Samples of PLA after 6 weeks are shown in Figure 2-11. While breakdown was most severe in highest irradiation dose samples, none of the samples were completely mineralized. Structural integrity Figure 2-12 shows views of irradiated/com posted PLA and reveals effectiveness of irradiation as a pre-composting step for enha ncing PLA breakdown. Structural integrity was affected and samples turned from clear to milky, smooth to porous, and glassy to powdery. The effect was more intense as irradiation dose in creased. Samples turned very brittle, showing sensitivity to even careful handling. During the composting process, PLA samples ar e subjected to high relative humidity and temperature. These conditions are favorable for h ydrolysis, where a reorganization of the smaller chain molecules causes an increase of the polym er crystallinity and opacity [34]. In addition, lower molecular weight polymers are more hydrophilic than corresponding high molecular weight polymers because of higher concentratio ns of hydrophilic end groups (both hydroxyl and carboxyl). Hydrophilicity affects osmotic pressu re causing influx of water into the polymer matrix, which causes a buildup of hydrostatic pressu re and consequent cracking and formation of 28

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microcavities. Microcacavitation occurs due to leaching of PLA material into the surrounding medium [35]. As long as hydrolysis produces small molecules of PLA, microbial activity will commence. Microorganisms begin to assimila te small molecules of PLA releasing carbon dioxide and water vapor, and weakening carbon to carbon bonds. It is presumable that the combination of these hydrolytic and biological eff ects is responsible for changes in structural integrity of PLA, turning it from glassy to porous and finally to powder. Weight loss Figure 2-13 shows results of weight loss of irradiated/composted PLA over time. Reduction of weight suggests metabolic conversi on of polymer in compost or into a soluble form. Greater irradiation doses and compost tim es resulted in increasing weight loss. For instance, un-irradiated PLA samples reduced weight by 1.3% after 10 weeks of composting whereas samples with absorbed doses of 216 kGy had reduced weight by 9.4%. This study exposed a critical aspect of PLA breakdown and compost behavior. Essentially, PLA requires relatively warm temper atures in order to soften a nd open the polymer structure to biological attack. As biologica l activity quickly progressed, a lo t of heat was released in a relatively short time. This time was too shor t to sufficiently convert PLA. Converters and manufacturers of PLA claim that biodegradation of the polymer takes place only at 58C for more than 6 weeks. Here, temperature was va riable and below 58C after the second week. After a few weeks in the composter, PLA samples with absorbed doses of 144 and 216 kGy turned very brittle and began to disintegrate from mechanical rotation of the co mposter. It is likely that these smaller pie ces, with larger surface area to vol ume ratios, and lower molecular weights due to irradiation, provi ded some advantage even as mi crobial activity diminished over time. 29

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Molecular weight Figure 2-14 shows how molecular weight of e-beam irradiated PLA was affected over composting time. Final weight-average molecula r weights of irradiated/composted PLA samples were 3.55x104, 1.86x104, 1.25x104 and 1.06x104 g/mol for the absorbed doses of 0, 72, 144 and 216 kGy, respectively. These values represent 21 %, 11%, 7% and 6% of initial un-irradiated PLA molecular weight. Therefore, even wh en breakdown was not totally achieved, it was demonstrated that e-beam irradiation is e ffective in improving degradation of PLA during subsequent composting. During the composting process, hydrolysis of polymers leads to molecular fragmentation, which can be regarded as a reverse poly-condens ation. This process starts with a water uptake phase followed by a splitting of ester bonds in a random way according to the Flory principle [33]. High relative humidity and temperature pr ovide conditions for cleavage of the ester linkages by water uptake and successive reduction in molecular weight [3, 36], as illustrated in Figure 2-15. After hydrolysis, microorganisms assimilate la ctic acid oligomers that may be soluble, releasing carbon dioxide and wate r. This two-step process demands reduction in PLA molecular weight early in the composting process. Neverthe less, rate of molecular weight reduction turned slower after the second week due to a reduction of microbial activity as evidenced by low temperature (35C instead of recommended 58C) and depletion of nutrients [20]. D-value Figure 2-16 shows D-value plots for molecula r weight versus dose after 0, 1, 2 and 6 weeks in the composter. Associated linear regres sion data and D-values are summarized in Table 2-1. Good linearity is observed in all plots with r-squares above 0.93. 30

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The D-values of irradiated PLA were about 430, 560, 380 and 410 kGy for 0, 1, 2 and 6 weeks of composting. Initial Dvalue suggests that ebeam irradiation treatment of 430 kGy on PLA will reduce its molecular weight in 90%. The a pparent increase in D-value (lower apparent sensitivity to irradiation) during the first week in compost may be due to chain recombination as compost temperatures increased above Tg. After the first week, D-values take their initial values, probably due to lower temperature. These results suggest that the primary e ffect of electron beam treatments on PLA composting behavior is the initial reduction of mo lecular weight. Irradiation essentially provides a head-start on PLA breakdown, but it does not app ear to significantly in crease sensitivity of PLA to hydrolysis during composting. Immersion of PLA in Alkaline Media Structural integrity Over time, it was noticed that PLA samples subjected to 1N NaOH fr agmented but without losing tensile properties. Samples remained hard and brittleness was fairly consistent. For both conditions (0.1N NaOH and 1N NaOH), edges of samples were observed to become rougher with concentration and time. Figure 2-17 shows magnified pictures (x10) to illustrate this phenomenon. Weight loss Samples of PLA immersed in 1N NaOH fragment ed over 22 days to such an extent that monitoring weight loss was not possible. For PLA samples immersed in 0.1N NaOH for 22 days, weight loss was around 14% as shown in Figure 2-18. Since brittleness was not affected, but weight dropped, it is believe d PLA dissolves under alkaline conditions, which is not the same as hydrolysis. Polymer dissolution is a consequence of molecular disentanglements and occurs more inte nsely on the surface than internally due to the 31

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direct contact with solvents in these areas. Th at phenomenon caused a heterogeneous sample in terms of entanglements. Molecular weight Exposure of PLA to alkaline media for 22 days did not significantly a ffect weight-average molecular weight (Figure 2-19). Pr esence of peaks ranging between 1.38x105 and 1.66x105 g/mol may be attributed to sample heterogeneity in terms of entanglements, which occur more at the surface rather than in the center. Thus, it may be possible that test samples were more representative of surface material, or vice versa, and affected the numerical results. It is important to recall that an assumption of the intrinsic viscosity method for molecular weight estimation is that entanglements are the same in the whole sample [37]. It appears that this assumption may not be valid in this case. Immersion of PLA in Acid Media Structural integrity Structure was not affected by a solution of nitric acid 0.1% or 0.016N (pH=1) after 22 days. There was no change in color, toughness, brittleness, weakness, or any other phenomena. This observation suggests that PLA is not vul nerable to acids at a temperature of 25C. Weight loss Dry weights of PLA samples did not change after 22 days. Absence of weight loss means that dissolution or reaction of PLA di d not occur in acid media at 25C. Exposure of PLA to Steam Structural integrity Samples of PLA exposed to steam for 4 and 8 hours displayed shrinking, brittleness and pore formation (Figure 2-20). These changes tended to be more severe as steam temperature and time increased. 32

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Shrinking is attributed to application of temp eratures above the glass transition temperature (Tg=58-70C) for PLA [10]. The glass transition is a second-o rder thermodynamic transition where polymers turn from glassy to rubbery st ate. In this state, change of volume with temperature is intensified and is revealed th rough shrinking and twistin g observed in samples. The mechanism of this phenomenon is transl ational motion of mol ecules and cooperative wriggling and jumping of segments of molecules, leading to flexibility and elasticity [37]. Increased brittleness was quite noticeable, and samples more intensely treated were more sensitive to subsequent handling. Samples gene rally exhibited cracks and broke easily. This suggests formation of amorphous regions caused by smaller molecules, rearrangement and rapid cooling. Pores formed during treatment are shown in Figure 2-21. This demonstrates severe disruption of PLA structure as well as exposure of greater areas of polymer that can be attacked during subsequent composting. The mechanism of pore formation was not determined, but is likely to be related to regional leaching of PLA to the surrounding medium as a result of hydrolysis, leaving cav ities or pores. Molecular weight Molecular weight analysis was conducted for PLA samples exposed up to 4 hours in steam. Beyond this time, molecular weight distributions were t oo broad for analysis. Figure 2-22 shows how PLA molecular weight was affected by steam treatments. Initial weight-average molecular weight was about 2.10x105 g/mol. After 4 hours at 100, 110 and 120C, samples achieved weight-average molecular weights of 6.00x104, 2.88x104 and 1.19x104 g/mol, respectively. These values represent about 29%, 14% and 6% of the initial molecular weight, for each respective treatment. 33

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Data reported by other authors show that po lyamide 11 subjected to high temperatures in acidified water (pH 4) reduces molecular weight by half in about 40 days at 100C, and 15 days at 120C [38]. Results here show that PLA exposed to similar treatments reduces molecular weight by half in about 2.2 and 0.5 hours, at each respective temp erature. While this comparison indicates that PLA is a good candidate for stea m hydrolysis, it also suggests that steam treatments may also serve as a means to separate PLA from other plastics in the waste stream. Dramatic decreases in molecular weight are the result of hydrolysis caused by high temperature and relative humidity, and can be re garded as a reverse pol y-condensation. Splitting of PLA ester bonds requires water and is helped with temperatur e, occurring in a random way according to the Flory principle [34]. More seve re treatments would involve more energy with sufficient moisture, yielding higher chain scission, and therefore, lower final molecular weights. The degree of chain scission was not determined for steam treatment, since crosslinking does not take place. Conclusions After evaluating different treatments to reduc e PLA molecular weight, it was concluded that exposure to steam is most rapid and eff ective. Samples of PLA treated for 4 hours with steam at 120C became extremely brittle and stea m caused weight-average molecular weight to decline by 94%. The mechanism of steam-treated PLA molecular weight reduction is basically thermal hydrolysis. Gamma irradiation is the second ranked tr eatment regarding PLA molecular weight reduction. Samples treated with 172 kGy -irradiation decreased molecular weight by 86%. Here, the main mechanism for this reduction is ra diolytic chain scissi on. Using electron beam irradiation, results were less satisfactory, which is likely due to protection from oxygen. In an 34

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additional experiment, e-beam irradiated PLA samples showed more sensitivity to composting conditions than untreated PLA samples, but even so, they did not achieve complete biodegradation. Use of alkaline and acid solutions to enhance PLA hydrolysis was not successful at the low temperatures applied. Alkaline solutions prom oted polymer dissolution, which might prove useful as part of a combined approach for accelerating PLA degradation. Table 2-2 summarizes molecular weight re duction results for each treatment. Given availability, familiarity and effectiveness of stea m treatment it is likely that steam would be the treatment of choice for accel erating PLA degradation. Figure 2-1. Drinking cups made of PLA Figure 2-2. Principle of intrinsic viscosity me thod for determination of molecular weight 35

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Dose (kGy) 020406080100120140160180200 Stress at break (MPa) 0 20 40 60 80 100 120 140 160 Figure 2-3. Stress at break of -irradiated PLA samples Dose (kGy) 020406080100120140160180200 Strain at break (%) 0 20 40 60 80 100 Figure 2-4. Strain at break of -irradiated PLA samples 36

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Dose (kGy) 020406080100120140160180200 M (g/mol) 0 2e+4 4e+4 6e+4 8e+4 1e+5 1e+5 1e+5 2e+5 2e+5 2e+5 Figure 2-5. Molecular weight of -irradiated PLA samples: Mw, Mv Dose (kGy) 020406080100120140160180200 1/Mn 1/Mn,o (mol/g) 0 1e-5 2e-5 3e-5 4e-5 5e-5 Figure 2-6. Determination of Gs in PLA -irradiation: Gs = 2.21 37

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Figure 2-7. E-beam irradiated PLA showing voids formation: (a) 0 kGy, (b) 72 kGy, (c) 144 kGy, (d) 216 kGy Dose (kGy) 0 50100150200250 M (g/mol) 2.0e+4 4.0e+4 6.0e+4 8.0e+4 1.0e+5 1.2e+5 1.4e+5 1.6e+5 1.8e+5 Figure 2-8. Molecular weight of e-beam irradiated PLA: Mw, Mv 38

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Dose (kGy) 0 50100150200250 1/Mn 1/Mn,o (mol/g) 0.0 2.0e-6 4.0e-6 6.0e-6 8.0e-6 1.0e-5 1.2e-5 1.4e-5 1.6e-5 1.8e-5 Figure 2-9. Determination of Gs in PLA e-beam irradiation: Gs = 0.52 30 35 40 45 50 55 60 65 70 0102030405060708T (C)Days0 Figure 2-10. Temperature profile of compost bulk 39

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Figure 2-11. E-beam irradiated PLA after 6 weeks in compost: (a) 0 kGy, (b) 72 kGy, (c) 144 kGy, (d) 216kGy Figure 2-12. Structural integrity of e-beam irradiated/composted PLA: (a) early cracks affecting surface, (b) layer formation and overall structure affected 40

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3.3 5.7 9.4 1.30 2 4 6 8 10 024681 0Weeksw (%)1 2 0 kGy 72 kGy 144 kGy 216 kGy Figure 2-13. Weight loss of ebeam irradiated/composted PLA 0 50000 100000 150000 200000 01234567Mw(g/mol)Weeks 0 kGy 72 kGy 144 kGy 216 kGy Figure 2-14. Molecular weight of e-beam irradiated/composted PLA 41

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Figure 2-15. Hydrolysis of PLA. 3.5 4.0 4.5 5.0 5.5 0 50 100 150 200 250Log MwDose(kGy) 0 wk 1 wk 2 wk 6 wk Figure 2-16. Plots of e-beam irradiation dose vs. log Mw for D-values estimation 42

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Figure 2-17. Structural integrity of PLA subjected to alkaline media: (a) 0.1N 2d, (b) 0.1N 22d, (c) 1N 6d, (d) 1N 13d 0 2 4 6 8 10 12 14 16 0 5 10 15 20 25Weight loss (%)Days Figure 2-18. Weight loss of PLA subjected to 0.1N NaOH 43

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90000 120000 150000 180000 0 5 10 15 20 25Mw(g/mol)Time (d) Figure 2-19. Molecular weight of PLA subjected to alkaline media Figure 2-20. Structural integr ity of steam-treated PLA: (a )100C-4h, (b)110Ch, (c)100Ch, (d)110Ch 44

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Figure 2-21. Pores in steam-treated PLA: 120Ch () 0 50000 100000 150000 200000 250000 012345Mw(g/mol)Time (h) 100C 110C 120C Figure 2-22. Molecular wei ght of steam-treated PLA 45

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Table 2-1. Linear regression outputs and D-values of e-b eam irradiated/composted PLA 0 wk 1 wk 2 wk 6 wk R-square 0.9322 0.9481 0.9426 0.9328 Slope -0.0023 -0.0018 -0.0026 -0.0024 D-value 427 564 384 412 Table 2-2. Comparison of treatments to reduce PLA molecular weight Treatment Mw reduction Conditions Av ailability (max 5) Gamma irradiation E-beam irradiation Alkaline hydrolysis Acid hydrolysis Steam 86 2% 65 9% <10% NA 94% 172 kGy 216 kGy NaOH 0.1N x 22d HNO3 0.1% x 22d 120C x 4h + + +++++ +++++ +++ 46

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CHAPTER 3 KINETICS OF REDUCTION OF MOLECULAR WEIGHT IN STEAM-TREATED PLA Introduction In the previous chapter, it was demonstrated that exposure of PLA to steam is the most effective treatment to reduce molecular weight. This chapter covers kinetics of reduction of molecular weight in steam-treated PLA. A first-order reaction m odel was proposed. This mathematical estimation would allow relating molecular weight with steam temperature and exposure time. In addition, this chapter conf irms that steam affects PLA through hydrolysis. Analysis of spectroscopy by Fourier Transform Infrared Attenuated Total Reflectance (FTIR-ATR) confirms depolymerization back to lactic acid. Methods First Order Reaction Model Molecular weight loss of PLA treated w ith steam at 100, 110 and 120C was assessed using first order kinetics. The mathema tical model follows Equation 3-1 where kT is the reaction rate constant at temperature T, and M is the molecular weight at time, t. Mk dT dMT (3-1) Solving the ordinary differe ntial equation with limits t = 0 to t, and M = Mo to M results in Equation 3-2: tk oTeMM (3-2) Kinetic constants, kT, were found to be correlated with absolute steam temperatures, T, in accordance to the Arrhenius behavior [39] shown in Equation 3-3. Activation energy, Ea, and pre-exponential factor, k0, were estimated by linearization. 47

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) RT E exp(kka oT (3-3) A final model for molecular weight of PLA, which is temperature and time dependent, is shown in Equation 3-4. This model allo ws prediction of molecular weight, M, of PLA with initial molecular weight, Mo, after exposure to steam at temperature, T, after time, t. ))exp(tkexp(M)tkexp(MMRT E 0 o T oa (3-4) Depolymerization of PLA to Lactic Acid Resulting from Steam Exposure An experiment was conducted in order to confirm that exposure of PLA to steam undergoes hydrolysis until complete depolymerizati on to lactic acid units. For this purpose, an extreme treatment of 120C for 24 hours was pe rformed. In this experiment, PLA samples (~30.1g) were placed in jars (hol ed lids) containing 100ml of de ionized water (pH 7.5) and then retorted. After steam treatment, jars contained residual solid PLA and a liquid drip, probably condensed water plus a byproduc t from PLA. Drip was collected and analyzed by pH (Accumet AR60, Fischer Scientific, Pittsbu rgh, PA) and FTIR-ATR (Nicomet 6700 Smart Orbit, Thermo Scientific, Inc) to confirm pr esence of lactic acid. Also, the yield of PLA conversion to its monomer lactic acid was determined (Equation 3-5). Variables Wo and Wf are PLA dry solid weights before and after the steam exposure for 24 hours. The term (Wo Wf) represents the weight of PLA that was converted to lactic acid. %100 W WW conversion %o fo (3-5) 48

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Results and Discussion First-order Reaction Model Figure 3-1 shows semi-log plots of molecu lar weight vs. time at 100, 110 and 120C. Slopes of curves, obtained by regression analysis represent the respectiv e kinetic constants, kT, and are shown in Table 3-1. Figure 3-2 shows temperature sensitivity of the reaction rate constants, kT, through the Arrhenius plot. Regression provide s estimates for activation energy, Ea, and Arrhenius preexponential factor, ko. Regression analysis yields an r-square of 0.996. Values for Ea and ko were 52.3 KJ/mol and -6.18x106 h-1. Other authors have found Ea of 51 KJ/mol for poly(L-lactic acid) (PLLA) in the melt at 180-250C [39] and 233 KJ /mol for PET in the melt at 250-280C [40]. This shows that activation energy for reduction of PLA molecular weight is much lower than that of PET, but similar to that for PLLA in a melt at higher temperatures. These values were used in Equation 3-4 to predict molecular weight reducti ons shown earlier in Figure 2-22. Figure 2-22 shows experimental and model predicted values. Depolymeryzation of PLA to Lactic Acid Resulting from Steam Exposure After exposure of PLA samples to steam at 120C for 24 hours, the solid mass was significantly reduced. This reduc tion suggested PLA conversion to a water-soluble compound in solution with condensed steam. Liqui d in the jar (drip) had a pH of about 1.5 indicating presence of acid, most probably lactic aci d. Finally, results of FTIR-ATR confirmed that the liquid byproduct was lactic acid. Figure 3-3 shows spectra for lactic acid from PLA and of DL-lactic acid control (Acros Organics, Geel, Belgium). These sp ectra appear to confir m liberation of lactic acid during steam treatment. As seen in the mass balance shown in Figure 3-4, weight of PLA decreased from 30.1g to 4.6g after this treatment. This represents a percentage of weight loss of 84.7% where a 49

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significant part converted to lac tic acid units. Tsuji et al. (200 3) studied melt hydrolysis of PLA and found a yield of L-lactic acid from PLLA of 90% at 250C for 10-20 minutes [39]. Ohkita and Lee (2006) investigated th e enzymatic hydrolysis of PLA using proteinase K and found a yield of lactic acid from PLA of 38% after 8 days at 37C [8]. Based on weight loss of PLA, it is presumable that yields of l actic acid here were not much different from those observed by hydrolysis in the melt. Conclusion It has been demonstrated that exposure to steam up to 120C is an excellent method to hydrolyze PLA. Treated samples became brittle and riddled with pores, and analysis performed by FTIR-ATR confirmed hydrolytic liberation of lactic acid to the treatment medium. Weight loss of PLA after 24 hours in steam at 120C wa s 84.7% and suggests significant conversion to lactic acid. A kinetic model describing molecular weight reduction in PLA as a consequence of hydrolytic steam treatments was presented. Degr adation followed first order kinetics with activation energy, Ea, of 52.3 KJ/mol. Predicted values fitted experimental data well. Availability of this model is very important because, if it is demonstrated that steam-treated PLA is affected under composting conditions, additional models may be developed to predict biodegradation behavior as a function of the st eam-treatment conditions. These simulations will ultimately be useful for optimizing the whole two-step process (pre-treatment and composting). Finally, this study shows that steam treatments may be suit able not only for making PLA waste more accommodating to commercial composti ng operations, but also, for assisting with separation of PLA from traditional plastic wastes. 50

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8 9 10 11 12 13 012345Time (h)ln Mw 100C 110C 120C Figure 3-1. First-order reaction plots for steam-treated PLA -1.50 -1.00 -0.50 0.00 2.50E-032.55E-032.60E-032.65E-032.70E-03ln(k)1/T (K-1)Ea= 52.3 KJ/mol Ko= 6177171 h 1 Figure 3-2. Arrhenius plot for steam-treated PLA 51

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Figure 3-3. Spectra of lactic acid obtained by FTIR-ATR PLA (30.1g) + water (100g) Figure 3-4. Mass balance of PLA treated with steam at extreme conditions Steam + water vapor Steam exposure 120C x 24h / condensation Drip (101.9g) PLA wet + water + lactic acid PLA wet PLA (4.6g) Drain Drying Steam Water vapor 52

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Table 3-1. Reaction rate constants, k (h-1), for molecular weight reductions of steam-treated PLA Temperature (C) 100 110 120 k 0.290 0.470 0.680 r2 0.975 0.989 0.988 53

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CHAPTER 4 DEVELOPMENT OF METHODS TO EVALUA TE AEROBIC BIODEGRADATION OF PLA Introduction The following standards are available for dete rmining aerobic biodegr adation of plastic materials under controlled composting conditions: American Society for Testing and Materials: ASTM D5338 International Standard s Organization: ISO 14855 These methods require mounting complicated systems involving temperature controlled vessels connected to permanent supply of oxygen and water vapor. Proposed vessels are too big (2-5 L) and temperature profiles are generated, providing variability in conditions inside the bulk. Additionally, heat generation can vary depending on compost weight and composition, which may also contribute to uneven temperature profiles. Figure 4-1 shows a diagram of the system configuration for ASTM D5338 [40], a nd it is required oxygen tanks, humidifiers, gas chromatographs, and baths / big ovens/ heaters. These issues, combined with monitoring duties, makes this method not practical for rapid biodegr adation assessments. A simpler approach would be desirable. This chapter investigates new ways to determine evolution of aerobic biodegradation instead of using standard methods. Aerobic biodegradation of PLA is measured by the ratio of carbon evolved as carbon dioxide (released during breakdow n), over initial carbon content. Mathematically, it can be expressed as Equation 4-1. % biodegradation = %100 PLAinitialincarbonofMass COevolvedincarbonofMass2 (4-1) If none of the carbon atoms in PLA molecule s converts to carbon in the generated CO2, biodegradation would be zero. In contra st, if all carbon is converted to CO2, then biodegradation would be 100%. Some carbon is believed to be converted to bicarbonates and carbonic acid, and 54

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other components may recombine with the humic acids present in the compost. Also, carbon from PLA may be assimilated into microbi al biomass. However, production of CO2 is the basis of biodegradation assessment s. Therefore, the capabil ity of quantifying evolved CO2 from the system is critical to any method de veloped to quantify biodegradation. In this chapter, three methods are desi gned, developed and assessed to measure PLA biodegradation evolution, including (1) the method of flexible bags, (2 ) the method of rigid containers, and (3) the method of perforated jars. The first two rely on principles of modified atmosphere packaging (MAP) and estimate CO2 production by combining a system molar balance and equations that govern gas permeability through plastic films. The third method relies on principles of gas diffusion and estimate CO2 production by combining a system molar balance and CO2 diffusion through holes in jars. Materials and Methods Method of Flexible Bags This method was proposed as a modification of the standard method ASTM D5338 [41] to overcome the issue of requiring permanent oxygen and water vapor supply. The use of a highly permeable film would permit oxygen transmission into the bag while withholding moisture content of the biomass. Thus, sele ction of the film is critical for this meth od. Ideally, this method requires a film that is very permeable to oxygen, impermeable to water vapor, and semipermeable to carbon dioxide. Unfortunately, no commercially available films meet these requirements, however high oxygen transmission rate (OTR) polyethylene was the closest to the requirements of this method, so it was used. Polyethylene film (Cryovac, Duncan SC) of 3.035mil thickness with high oxygen transmission rate (9224 cc m-2d-1 @ 25C) was used to prepare bags with dimensions 9cm x 12cm. A thermosealer (Sencorp Systems model 12SC/1) was used for sealing edges. Rubber 55

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pads (sticky nickels, Mocon, Inc., Minneapolis, MN) were glued on their surfaces to serve as syringe needle ports injecting water or taking ga s samples from the headspace. Bags were filled with 40g of 3-month mature compost (Appendix A) and 8g of PLA. Control bags included only 40 g of compost. Filled and sealed bags were stored in a Lab-Line L-C Incubator (Lab-Line Instruments, Inc., Melrose Park, IL) at 58C fo r 40 days. Figure 4-2 shows a diagram of the flexible bags and the main interactions between the biomass (compost + PLA), the headspace and the environment, and pictures can be seen in Figure 4-3. Periodically, bags were agitated, water was in jected (~1-3ml) and gas samples from the headspace were taken for analysis. Headspace gas analysis was performed using a dual headspace analyzer (Pac Check 650, Mocon, Inc., Minneapolis, MN) to determine carbon dioxide and oxygen concentration ov er time. A molar balance of ca rbon dioxide in the system is expressed in Equation 4-2. Oxygen and nitrogen concentrations we re not used for this method since they are not a carbon source. CO2 generated = CO2 permeated + CO2 headspace (4-2) The number of moles permeated can be es timated by Equation 4-3, derived from the definition of permeability [42]. E tp]CO[AP E tpAP permeated COatm2 2CO CO2CO 22 (4-3) The number of moles in the headspace can be estimated by Equation 4-4, derived from the universal gas law [43]. R T V]CO[p R T Vp headspace COhs2 atm hs2CO 2 (4-4) 56

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Film permeability to carbon dioxide, PCO2, at 58C was estimated using the methodology described in Appendix B and was equal to 13.41 mol.mil / (atm.d.m2). The volume of the headspace, Vhs, was estimated by subtracting the volum e occupied by the compacted biomass (Appendix C) to the bag volume (Appendix D) which was measured with a volume-meter designed and constructed for this purpose. Production of CO2 solely from PLA is the difference between CO2 produced from the mixture PLA-compost and CO2 produced by the compost itself (control). Once it is known how many moles of CO2 are due to PLA, the carbon mass can be determined by multiplying the number of moles by 12 which is the molecular weight of carbon. Finally, total biodegradation is the ratio of carbon mass evolved as CO2 to initial carbon mass in PLA sample assessed. Weight of carbon in PLA is half its total weight (Figure 4-4). To organize and simplify calculations, Table 4-1 was developed. Plots of columns (1) and (10) provide PLA biodegradation. Method of Rigid Containers with Plastic Film Lids A cylindrical, rigid, wide mout h container with 2850cc capacity was adapted with ports, filled with 100g of 4-month mature compost (A ppendix A) and 10g of PLA. Control was filled with only 100g of compost. Mouths of containers, with internal diameters of 15cm, were covered with high OTR polyethylene film (Cryovac, Duncan SC) (thickness ~3.035mil, OTR ~9224 cc/(m2.d) @ 25C) that served as the material to allow O2 and CO2 permeation. Containers were stored in a Lab-Line L-C Incubator (Lab-Line Instruments, Inc., Melrose Park, IL) at 58C for 18 days. Figure 4-5 shows a diagram of the rigid c ontainers with plastic lids, as well as the main interactions between biomass (compost + PLA) headspace and environment. A picture of the system is shown in Figure 4-6. 57

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Daily, containers were agitated, water was injected to maintain proper moisture (~4555%), and gas samples from the headspace were take n for analysis. Also, for better performance, oxygen was injected daily (~100 300cc) to maintain aerobic conditions. The headspace was analyzed in a dual head space analyzer (Pac Check 650, Mocon, Inc., Minneapolis, MN) to determine carbon dioxide and oxygen concentrations. Over time, data for carbon dioxide concentrati on in the headspace were collected. As in the method of flexible bags, the number of moles of CO2 generated by the biomass is the sum of the moles permeated out of the container and the mo les in the headspace. Expressions that govern each component of this molar balance are Equati on 4-3 and Equation 4-4 but with the new data from this experiment. Thus, Table 4-1 was also us ed to process these data and plots of columns (1) and (10) provided evolution of PLA biodegradation assessments. Method of Perforated Jars Glass mason jars of 936cc of capacity were filled with 100g of 5-month mature compost (Appendix A) and 10g of PLA. Controls were f illed with only 100g of compost. Lids were drilled with five 1/16 holes to allow gas tran sfer between the environment and jars headspace. Closed jars were st ored in a Lab-Line L-C Incubator (Lab-Line Instruments, Inc., Melrose Park, IL) at 58C for 34 days. Figure 4-7 shows the diagram of perforated jars and the main interactions between biomass (compost + PLA), headspace and environment. Pictures of a jar filled with 1-week biomass and arrangement of 5-holes is shown in Figure 4-8. Daily, jars were agitated a nd concentration of oxygen and ca rbon dioxide in the headspace were determined using a dua l headspace analyzer (Pac Check 650, Mocon, Inc., Minneapolis, MN). Periodically, water (~2-4cc) was added th rough the holes to compensate for water loss. A molar balance of carbon dioxide in the system is as follows (Equation 4-5): CO2 generated = CO2 diffused + CO2 headspace (4-5) 58

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The number of moles, diffused represents the average of moles flowing through the holes. This value can be estimated by Equation 4-6, derived from Ficks first law that states that the flux or rate of transport of an ideal gas is linearly related to its concentration gradient [42]. t]CO[D diffused CO2ef 2 (4-6) The number of moles in the headspace can be estimated by Equation 4-7 which is derived from the universal gas law [43]. RT V]CO[p RT Vp headspace COhs2 atm hs2CO 2 (4-7) The overall coefficient of diffusion, Def, was determined in a parallel experiment (Appendix E). The volume of the headspace was es timated by subtracting the volume occupied by the biomass (Appendix C) from the total volume of the jars which was 936cc. Production of CO2 solely from PLA is the difference between CO2 produced from the mixture PLA-compost and CO2 produced from the compost itsel f (control). The carbon mass can be determined by multiplying the number of moles of CO2 produced by 12, which is the molecular weight of carbon. Finally, total biodegr adation is the ratio of carbon mass evolved as CO2 to initial carbon mass of PLA samples assessed. Initial weight of carbon in PLA samples is half the sample weight based on the molecular formula of PLA. To organize and simplify calculations, Table 4-2 can be completed. Plots of columns (1) and (10) provide PLA biodegradation. Results and Discussions Method of Flexible Bags The method of flexible bags was develope d to simplify analysis of biodegradation behavior. Ideally, this method could eliminate th e need for continuous gas flow and moisture 59

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management. To sustain aerobic composti ng conditions the film requires high OTR characteristics. The film chosen has the highest OTR of any readily available commercial film. At composting conditions, the film proved to be too fragile to manipul ate. Additionally, the films water vapor transmission rate (WVTR) proved to be higher than desirable for this application. However, the theory supporting th e approach is sound a nd this analysis and summary are provided as a means fo r suggesting future work if a nd when a better film becomes available. Main issues encountered during the ex periment performed with the method of flexible bags follow: Moisture loss and related punctures: Water va por permeated out of the bag faster than expected, so moisture content of biomass dropped quickly. This problem was solved by injecting water into bags more frequentl y, but this required additional punctures and resultant leaks. Abrasion of film: Shaking of the bags, in tended to homogenize internal conditions (oxygen supply, heat and moisture), caused ab rasion and damage to the film. This may have created small holes and l eaks that were not visible. Strength loss of film: Formation of by-pr oducts from PLA biodegradation (i.e. lactic acid) reduced the pH of the biomass, and in concurrence with temperature and high relative humidity, deteriorated the film surf ace. Film developed a burn-like appearance and lost strength. Modification of film properties could affect permeability of the film to CO2. Oxygen depletion: When biological activity bega n to take place, samples started to show elevated rates of oxygen consumption. This fact depleted the oxygen supply in the headspace and turned the process anaerobi c. ASTM D5338 recommends oxygen levels above 6% for ensure aerobic conditions [41] and some of the experiments got 0% oxygen. This issue could have been overcome by increasing the film area. However, something unexpected was found; even wit hout oxygen, CO2 genera tion still increased. This suggests that facultative microorganisms were active and may have been responsible for biological activity with or without oxygen. Figure 4-9 shows a picture of a bag in its day 40th that after many days under anaerobic conditions, showed total disappearance of PLA. Results of untreated PLA biodegradation, obta ined using this met hod, are shown in Figure 4-10. Large standard deviations were found among the 5 replications performed. For example, after 40 days one sample was 7.5% biodegraded whereas another was 30.3%. This disparity was 60

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primarily due to issues related to the film used. Thus, this method using currently available high permeation film is not sufficiently reliable. Method of Rigid Containers with Plastic Film Lids The method of rigid containers with plastic film lids was designed to overcome the issues encountered in the method of flexible bags. The design involved ports attached to the walls of the rigid container in order to avoid damaging the delicate film. Also, since the plastic film served as the containers lid (d id not contact the biomass), abra sion and strength loss issues were eliminated. To avoid oxygen and water vapor depletion, gases were injected daily to maintain required ranges (O2 between 10-21%, moisture between 45-60%). An unavoidable issue was the plastic lid defo rmation caused by the water vapor pressure, and its consequent effect in th e permeability estimation. Deformati on of the plastic film lid was measured and estimated to add 5%. After 18 days in rigid containers with plas tic film lids, PLA biodegradation was estimated to be 10.1% as seen in Figure 4-11. As with e xperiments of the flexible bags, the trend was almost constant during the first week and then increased at a constant rate. It is believed that in the presence of PLA, microorga nisms slow down biological activ ity in the surrounding organic matter (compost) in order to adap t to the new source of carbon. This method overcomes most of the issues enco untered in the method of flexible bags, but still left room for improvement. Remaining issues are that CO2 permeability may be affected by water vapor as well as issues with variable geometry due to st retching. These observations led to a new method that avoided plastic film. Method of Perforated Jars The method of perforated jars did not present the issues enco untered in previous developed methods. The problems of area, volume and leaking of the system were eliminated by the rigidity 61

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of the container, and water vapor pressure eff ects were avoided by presence of holes. The holes served not only to allow mass transfer (O2, CO2), but also to inject wate r or take gas sample from the headspace. The overall effective co efficient of diffusion (5 holes) for CO2 was found to be Def = 0.00031 mol/h/% and was estimated by the slope of the plot of experimental calculated effusion rate vs. %CO2 shown in Figure 4-12. The r-square value obtained by linear regression analysis was found to be practically 1, indicating an almost perfect linear dependence of effusion rate with CO2 concentration inside the jar, as expected. In Figure 4-13, PLA biodegrada tion was obtained for a remaining solid sample (no drip) that was exposed to steam at 100C for 4 hours. Higher rates of biodegrad ation were observed in these samples compared with those of untreat ed samples assessed using the method of rigid containers with plastic film lids. Conclusion Among the methods developed and assessed to perform PLA biodegradation, the method of perforated jars was found to be the most re liable and simple. It does not require mounting a system as the one proposed in the standards (ASTM D5338 and ISO 14855) that need permanent oxygen and water vapor supply. This method is much easier to conduct and adapt. For example, if it is expected that biological consumption of oxygen will be much faster, more holes could be added. The only drawback of this method is the requirement to inj ect water about every two days to maintain moisture content in the proper ra nge. In this regard, a recommendation could be integration of a water dispenser to the jars in order to recover water released through holes as water vapor. The method of flexible bags a nd rigid containers with plastic film lids had too many issues, the most significant being the possible alterati on of the film due to high relative humidity, temperature and vapor pressure in the system This issue can be overcome by determining a 62

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dynamic permeability coefficient of the film to carbon dioxide, which would be a function of temperature, and relative humidity and vapor pressure as well. Identifying a film that fulfills requirements of gas transmission rates (CO2, O2 and WVTR) is also difficult at this time. Figure 4-1. Set up for plastic biodegradat ion assessment in compost ASTM D5538 Figure 4-2. Interactions in flexible bags 63

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Figure 4-3. Pictures of flexible bags with biomass biodegrading Figure 4-4. Chemical formula of PLA 64

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Figure 4-5. Interactions in rigid container with plastic film lid Figure 4-6. Picture of rigid container with plastic film lid 65

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Figure 4-7. Interaction in perforated jar Figure 4-8. Picture of jar filled wi th biomass and perforated lid 66

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Figure 4-9. Picture of compost plus PLA after 40 days, in and out of the bag Days 01020304050 % biod. -10 0 10 20 30 40 Figure 4-10. Biodegradation of PLA us ing the method of flexible bags 67

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-2 0 2 4 6 8 10 12 0246810121416182% biodegradationDays0 Figure 4-11. Biodegradation of PLA using the met hod of rigid containers with plastic film lids 0.0000 0.0002 0.0004 0.0006 0.0008 0.0010 0.0012 0.0014 0.0016 0.0018 0.0020 01234567Effusion rate (mol /h)% CO2 Def = 0.00031 mol/h/% Figure 4-12. Overall effective coefficient of diffusion for CO2 through 5 holes 68

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-10 0 10 20 30 40 50 60 70 02468101214161Days% Biod.8 Figure 4-13. Biodegradation of PLA us ing the method of perforated jars Table 4-1. Biodegradation of PLA by method of flexible bags (1) Time, t (2) [CO2] (3) CO2 permeated (4) CO2 permeated accumulated (5) CO2 in headspace Determined in headspace analyzer Use Eq.4-3 (3)t=i + (4)t=i-1 Use eq.4-4 Table 4-1. Continued (6) CO2 total produced (7) CO2 total produced by control (8) CO2 total produced by PLA (9) Grams of carbon in CO2 from PLA (10) % biodegradation (4) + (5) Follow (1) to (6) for bags with only compost (6) (7) 12 (8) (9)*100 /0.5*WPLA Table 4-2. Biodegradation of PLA by the method of perforated jars (1) Time, t (2) [CO2] (3) CO2 diffused (4) CO2 permeated accumulated (5) CO2 in headspace Determined though headspace analyzer Use Eq.4-6 (3)t=i + (4)t=i-1 Use eq.4-7 69

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Table 4-2. Continued (6) CO2 total produced (7) CO2 total produced by control (8) CO2 total produced by PLA (9) Grams of carbon in CO2 from PLA (10) % biodegradation (4) + (5) Follow (1) to (6) for bags with only compost (6) (7) 12 (8) (9)*100 /0.5*WPLA 70

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CHAPTER 5 BIODEGRADATION OF TREATED PLA UNDER ANAEROBIC CONDITIONS Introduction Although the goal of post-consumer use of PLA is to return to th e environment during composting, this is not currently happening. Separating PLA from waste and acceptance of PLA in composting plants are proving problematic. In stead, PLA waste is being sent to landfills, where anaerobic conditions prevail and it appears that PLA does not biodegrade significantly [9]. Little is known about the fate of PLA in landf ills. Therefore, an objec tive of this study was to evaluate PLA degradation under anaerobic conditions. Under anaerobic conditions, organic matter usually degrades in four stages: (a) hydrolysis, (b) acidogenesis, (c) acetogenesis, and (c) methanogenesis [44, 45]. During hydrolysis, molecu les split and become smaller and soluble resulting in conversion of carbohydr ates, fats and proteins, into sugars, fatty acids and amino acids. This chemical reaction requires water and is aided by temperature and enzymes. Later, during acidogenesis, simpler compounds unde rgo fermentation carried out by acidogenic bacteria, and produce volatile fatty acids, hydrogen and carbon dioxide. Later, during acetogenesis, acetic acid, hydrogen and carbon dioxide are produced. During methanogenesis, the final products of the anaerob ic digestion are obtained. These are methane and carbon dioxide. For this work, it was postulated that untreated PLA biodegrades at a very slow rate because of its large molecular weight, ma king the necessary first step of hydrolysis difficult. However, pretreatments with steam or irra diation may assists with and/or substitute for hydrolysis and therefore allow PLA to be convert ed. It is supposed that main anaerobic reactions for converting PLA to biogas follow the sequence given in Figure 5-1. It is suspected that the amount of each intermediate product depends on the microbial strains and temperat ures of incubation. 71

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Experiments performed include weight loss of irradiated PLA (gamma and e-beam) and biochemical methane potentia l (BMP) of steam-treated PLA (120C x 0h, 3h), both under mesophilic and thermophilic conditions. Also, an experiment of untreated PLA in DI water in absence of oxygen was conducted to assess thermal hydrolytic degradation by itself. Materials and Methods Weight loss of PLA in Water under Anoxic Conditions Rectangular sheets of PLA (1cm x 4cm x 0.2mm) were placed in gla ss bottles (cap 280ml) containing 100ml of DI water. Jars were flushed with gas N2/CO2 (70/30) for 30 minutes to remove oxygen and then immediately sealed. Some ja rs were stored for 180 days at 37C in an IsotempTM Low Temperature Incubator (Fisher Scientif ic, Inc., Philadelphia, PA). Others, at 58C in a Lab-Line L-C Incubator (Lab-Line Instruments, Inc., Melrose Park, IL) for the same time. Dried and cleaned PLA samples were we ighted at the beginning and the end of the experiment to evaluate their thermal degrad ation in absence of oxygen at 37C and 58C. Weight Loss of Irradiated PL A in Anaerobic Biological Media Clear glass bottles (cap. 280ml) were filled wi th small pieces of irradiated PLA (~5mm x 7mm x 0.2mm), 90 ml of nutrient formula (Appendi x F) and 10 ml of inoculum. Irradiated PLA pieces were subjected to gamma rays from ces ium 137 source (0, 72 and 172 kGy) and electron beam (0, 72, 144 and 216 kGy). Controls consiste d of media and inoculum but without PLA. Inoculum was provided by the Bioprocess Lab of the Agricultural & Biological Engineering Department at University of Florida. Inoculum was cultures of naturally occurring microorganisms, capable of growing under mesophilic (25-45C) or thermophilic (>45C) conditions [19]. After bottles were f illed, they were flushed with gas N2/CO2 (70/30) for 30 minutes to remove oxygen. Bottles were sealed and stored for 180 days at 37C in a IsotempTM Low Temperature Incubator (Fisher Scientific, In c., Philadelphia, PA), and at 58C in a Lab72

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Line L-C Incubator (Lab-Line Instruments, Inc ., Melrose Park, IL). These temperatures provided favorable conditions for biologica l activity of mesophilic and thermophilic microorganisms, respectively. Weight of dried and cleaned PLA samples was determined at the beginning and end of the experiment to evaluate effects of irradiation in anaerobic biodegradation. Bottles that were set up looked like in Figure 5-2. Biochemical Methane Potential (BMP) of Steam-Treated PLA in Anaerobic Biological Media The biochemical methane potential 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 provi des a simple means to monitor relative biodegradability of substrates. Clear glass serum-bottles (cap. 280ml) were filled with 1 gram of ground solid steamtreated PLA, 100 ml of nutrient formula (Appendix F) and 10 ml of naturally occurring inoculum (mesophilic or thermophilic) supplied by the Biop rocessing Lab of the Agricultural & Biological Engineering Department at University of Flor ida. Grinding of PLA was performed using an Urschel 3600 grinder (Urschel Laboratories, Inc., Valparais o, IN) with a 3mm screen. Ground PLA was a sample of the solid residue after steam treatment (120C x 0h and 3h). Drip was discarded. Controls consisted of glass bottles with the nutrient formula and inoculum, but without PLA. After filling, bottl es were flushed with gas N2/CO2 (70/30) for 30 minutes to remove oxygen. Later, bottles were sealed a nd stored for 28 days at 37C in a IsotempTM Low Temperature Incubator (Fisher Sc ientific, Inc., Philadelphia, PA ), and at 58C in a Lab-Line LC Incubator (Lab-Line Instruments, Inc., Melr ose Park, IL). These temperatures provided favorable conditions for biological activity of mesophilic and thermophilic microorganisms. 73

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Biochemical methane potential bottles and steam-treated grou nd PLA looked as in Figure 5-2 and Figure 5-3. Weekly, gas production and composition were determined using a Gas Partitioner Chromatograph model 1200 (Fisher Scientific, In c., Philadelphia, PA) ad apted with a thermal conductivity detector. Biochemical methane poten tial (BMP) of PLA was expressed as yield of methane per gram of PLA sample loaded into BMP bottles, and was determined in accordance to ASTM E1196 [46]. Data collected were inserted into columns (1), (2) a nd (3) of Table 5-1. The volume of methane removed was calculated and expr essed at standard cond itions of temperature and pressure (0C, 1atm) after re moving the vapor pressure effect at respective temperatures. Plots of columns (1) and (11) de pict the yield of methane per gr am of PLA (untreated and steamtreated). Results and Discussion Weight Loss of PLA in Water Under Anoxic Conditions Table 5-2 shows the weight loss of untrea ted PLA in oxygen-free water after 180 days. Results demonstrate that degradation occurs at 58 C but not at 37C. Evaluated samples were not subjected to any kind of chemical reaction or biological activity, so mechanism for degradation in absence of oxygen was solely thermal hydrolysis. Weight Loss of Irradiated PL A in Anaerobic Biological Media Under thermophilic conditions (58C), all samples were disintegrated and lost in the media. It can be said that we ight loss was almost 100%. In some cases, gelatinous PLA pieces were found which easily dissolved in the media when subjected to agitation. At this temperature, the mechanism of PLA degradation is a combin ation of thermal hydrolysis and biological activity that would produce methane. These data were not useful for determining effects of 74

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irradiation dose, irradiation source and biologi cal activity on PLA, so data collected from mesophilic conditions were used for this purpose. Figure 5-4 shows weight loss of irradiated PLA (gamma and e-beam) under mesophilic conditions (37C) after 180 days. PLA samples appeared to be much more vulnerable to anaerobic biological media when pre-irradiated with gamma source rather than e-beam. For instance, -irradiated PLA at 172 kGy lost 45% of wei ght whereas e-beam i rradiated PLA at 216 kGy lost only 3% under same anaerobic biologica l conditions after 180 days. This difference likely occurred because gamma irradiated PLA sa mples had lower molecular weights and weaker structures than those irradiated with electron beam (see Figure 25 and Figure 2-8), so they were more sensitive to biological activity. At 37C, un-irradiated PLA samples presente d negligible weight loss under anaerobic conditions. But, as seen in Figur e 5-4, irradiation dose played an important and accelerated role (more when treated with gamma rays) in PLA anaerobic biodegradation. That means that at higher irradiation doses, the we ight loss effect was much more pronounced than at lower irradiation doses. At this temperature, the main mechanism for weight loss is biological activity performed by mesophilic microorganisms, since ther mal hydrolysis has been shown not to occur. Biochemical Methane Potential (BMP) of Steam-Treated PLA in Anaerobic Biological Media Figure 5-5 shows methane production by untreat ed and steam-treated PLA under anaerobic conditions. Untreated PLA subjected to mesoph ilic conditions did not degrade. However, samples subjected to thermophilic conditions did degrade. Therefore, temperature of incubation is a key factor for anaerobic biodegradation of PLA. Results obtained for mesophilic conditions match data from literature [4, 47]. Untreated PLA subjected to thermophilic conditions produced 187 ccCH4/g in 56 days. No data has been found in literature regarding PLA degradation under 75

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thermophilic conditions. In this work, it was demonstrated that PLA evolves methane after 21 days in anaerobic media at 58C. This result was anticipated from prior work with compost when conditions unintentionally became anaerobic, but yet CO2 production at 58C was observed. For these samples, hydrolysis and acidification took place mostly during the first 3 weeks, followed by methanogenesis. Regardless of temperature of incubation, steam-treated PLA samples (120C x 3h) produced more methane than untreated PLA. Under mesophilic and thermophilic conditions, yields of 90 and 225 ccCH4/g were obtained for treated PLA (improving negligible and 187 ccCH4/g from untreated). At 58C, steam-treated PLA began producing CH4 at the beginning of incubation, whereas for untreated PLA, CH4 was not observed until after the third week. This suggests that steam treatment provides a head st art effect. At 37C, it was shown that PLA can biodegrade in anaerobic media only if material is pretreated. Conclusions Untreated PLA does not degrade under mesophilic conditions, but does under thermophilic conditions. Methane yield of 187 ccCH4/g after 56 days at 58C was observed. Mechanism for degradation is hydrolysis and aci dification during the first 3 week s, followed by methanogenesis. Treated PLA, using steam or irradiation, does biodegrade under anaerobic conditions, either in mesophilic or thermophilic circumstances. This means that it will ultimately evolve as carbon dioxide and methane at either temperat ure range. For steam-treated PLA (120C x 3h), methane production was 90 and 225 ccCH4/g after 56 days of incubation at 37C and 58C. Gamma irradiated PLA samples biode graded faster than e-beam irra diated PLA, which is related to oxygen protection afforded by high dose rates associated with e-beam treatments. Gamma treatment was more effective in reducing molecula r weight, which contributed to the head start effect of -radiation. Gamma irradiated PLA with absorbed dose of 172 kGy lost 45% of its 76

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weight at 37C after 180 days. This reducti on in weight is main ly caused by biological conversion of PLA to aqueous soluble oligomers, lactic acid, acetic acid, and probably evolution to carbon dioxide and methane. Also, it was noticed that regardless of ir radiation source (i.e gamma irradiation/Ce 137 or e-beam), highe r absorbed doses provided higher rates of biodegradation. Figure 5-1. Supposed anaerobic reactions for PLA anaerobic biodegradation Figure 5-2. BMP bottle with anaerobic media Figure 5-3. Steam-treated ground PLA 77

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0 10 20 30 40 50 60 0 50 100 150 200 250Dose (kGy)Weight loss (%) Gamma 37C E-beam 37C Figure 5-4. Weight loss of irradiated PLA 225 187 90 2-20 20 60 100 140 180 220 260 300 340 380 420 460 01 02 03 04 05 06dayscc CH4 (STP) / g0 Thermophilic treated PLA Thermophilic untreated PLA Mesophilic treated PLA Mesophilic untreated PLA Theoretical Figure 5-5. Conversion of steam-t reated PLA (120C x 3h) to CH4 under anaerobic conditions 78

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Table 5-1. Production of methane in steam-treated PLA (1) Time (2) cc gas removed (3) % CH4 (4) cc CH4 removed (5) cc cumulative CH4 removed (6) cc CH4 in headspace (7) cc CH4 total Syringe displacement GC (2)*(3)/100 (4)t=i + (5)t=i-1 Vhs*(3)/100(a) (5) + (6) Table 5-1. Continued (8) cc CH4 total @ STP (9) cc CH4 total produced by control (10) cc CH4 total produced by PLA (11) CH4 yield (cc/ g VS) (7)*(273.15/T)*F(b) Follow steps (1) to (8) for bottles with control (8) (9) (10)/WPLA (a) Estimated in each gas sample analysis (b) Correction due to pressure vapor: F=(760-pvap)/760. pvap(58C) = 136mm Hg. pvap(37C) = 47mm Hg [48] Table 5-2. Weight loss of untreated PLA in water under anoxic conditions Temperature (C) Weight loss (%) 37 58 0.190.2 98.961.5 79

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CHAPTER 6 BIODEGRADATION OF STEAM-TREATED PLA UNDER COMPOSTING CONDITIONS Introduction Reactions that occur during PL A biodegradation in a compos ting process occur in three stages summarized in Figure 6-1. First, PLA hydrol yzes producing lower molecular weight PLA. This stage requires water and energy, but the pres ence of microorganisms is not essential. Then, low molecular weight PLA under goes production of oligomers and lactic acid. During this second stage, hydrolysis still occu rs but biological activity takes place more intensively, and is aided by appropriate temperature, and moisture and oxygen levels. The third stage is carried out only by biological activity and produces carbon dioxide and wa ter [36]. Depending on the pH and the microbial cultures, radica ls could also be produced and combined with the biomass to integrate humic acids in the compost. Also, a sm all part of the carbon dioxide fraction transforms to carbonic acid and bicarbonates transformations due to high mois ture levels in the media. The main objective of this research is to eval uate the effectiveness of different processes as potential pre-composting treatments for PLA waste, and to determine whether they will allow complete degradation within the time frame of normal organic compost. In Chapter 2, it was demonstrated that exposure of PLA to steam is the most effective treatment to reduce molecular weight and affect st ructural integrity. In this chapter, steam-treated PLA samples were subjected to co mposting conditions to evaluate their biodegradation, via the method of perforated jars de scribed previously (Chapter 4). Additional experiments to determine the ki netics of biodegrada tion were performed. Results of this work validate the hypothesis of this study, which states that pre-composting treatments able to reduce PLA molecular weight will be favorable in subsequent composting processes by reducing overal l biodegradation time. An important question is whether 80

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pretreatments provide a head start in further composting, and/ or contribute to accelerate conversion of PLA. The head start effect is shown in Fi gure 6-2. A typical curv e representing PLA biodegradation in the composting process can be described through 3 phases: (a) a lag period, (b) an accelerated biodegradation phase and (c) a decelerated biodegr adation phase until reaching a plateau. A head start effect would shift the cu rve in time, so that the lag period would be shortened or eliminated, but the trend of the curv e would be maintained. So, a head start effect would be expected to displace the entire curve to the left. As a consequence of this head start effect, overall biodegradation time will be reduced. The acceleration effect is illu strated in Figure 6-3. Here, the biodegradation rate must be carefully analyzed once the lag pe riod is complete. The slope of the curve (in the earlier phase) represents initial biod egradation rate, and is an indicator of how ra pidly carbon dioxide is evolving. In Figure 6-3, the dashed curve depicts biodegradation e volution of PLA exhibiting the acceleration effect, represented by a steeper slope. Material and Methods Steam-Treated PLA Biodegradation in Compost Previously (Chapter 4), the me thod of perforated jars was de scribed. In this section, this method was used to determine the kinetics of steam-treated PLA biodegradation in compost. Samples of PLA were ground using an Urschel 3600 grinder (Urschel Laboratories, Inc., Valparaiso, IN) with a 3mm screen, and then subjected to steam at 120C for 0, 1, 2 and 3 hours. Mason jars of 936cc capacity provided with 5 ho les (x 1/16) in the lids were filled with 100g of 6-month mature compost (Appendix A) and 10g of ground PLA samples. Sealed jars were stored at 58C fo r 31 days in a Lab-Line L-C Incubator (Lab-Line Instruments, Inc., Melrose Park, IL). Beside routine practices such as agitation and moistu rizing, concentration of 81

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gases in the headspace was determined daily using a gas analyzer Pac Check 650 (Mocon, Inc., Minneapolis, MN). Data collect ed was processed according to the methodology described earlier in chapter 4 in order to create curv es of PLA biodegra dation over time. Kinetics of Steam-Treated PLA Biodegradation in Compost Data of biodegradation were plotted and ad justed to the logistic model with three parameters shown in Equation 6-1. Parameters were estimated using nonlinear regression performed with SigmaPlot v.10. Ideally, parameter a should be 100. Parameter b is associated with the lag period, and the parameter xo represents the time at whic h half of the biodegradation would be completed. For untreated PLA, large values of parameters b and xo were expected, whereas for treated PLA smaller values were expected. b o)x/t(1 a .biod% (6-1) Weight Loss of Steam-Treated PLA in Compost and Comparison with other Common Feedstock Flat sheet samples with rectangular or circ ular shapes, with similar surface area (~12.5 cm2) were prepared from steam-treated PLA wr apped in nylon screen envelopes, wood and virgin corrugated paperboard. Th ese samples were dried, weighed and immersed in water for 10 minutes. Wet samples were placed individually into perforated mason jars (cap.936cc) containing 200g of 6-month mature compost (Appe ndix A). Controls were jars filled with 200g compost. Closed jars were stored in a Lab-Line L-C Incubator (Lab-Line Instruments, Inc., Melrose Park, IL) for 14 days at 58C. Periodically, jars were gently shaken to ensu re uniform contact of samples with compost, and water was injected to maintain proper moisture content of the biomass. Samples were covered by the compost at all times to promote bi ological activity. At the end of the experiment, 82

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samples were removed from the jars, carefully wa shed, dried and weighted. Weight loss of each individual sample was determined using Equation 2-1. Results and Discussion Steam-Treated PLA Biodegradation in Compost Figure 6-4 shows evolution of steam-tre ated PLA (120C x 0, 1, 2 and 3 hours) biodegradation under composting conditions. On aver age, rates of biodegradation increased as steam treatment became more severe. These resu lts validate the general hypothesis of this study, which states that a pre-composting treatment cap able of reducing PLA molecular weight would increase biodegradation rate in subsequent comp osting process. This figure also confirms the head start and acceleration effects, whic h were postulated as means by which PLA conversion would be enhanced. As steam treatment increased, head star t and acceleration effects also increased. During the experiment, oxygen concentration in the headspace was monitored and found to be above 17.8% at all times. Agitation permitte d good aeration and mixing, but it needed to be performed carefully in order to minimize clum ping of the particles. Unfortunately, clumping occurred in the jar containing PLA treated with steam for 3 hours at 120C during the last days of the experiment. Clumping appeared to slow biodegradation during thos e last days, probably due to a lesser area expo sed to the environment. According to standards, biodegradabililty requires 60% conversi on. In this regard, samples treated at 120C for 3, 2 and 1 hours re ached biodegradability after 14, 16 and 19 days. Untreated samples did not achieve biodegradability even after 31 days. Observations of biomass through glass jars confirmed previous results. Steam-treated PLA at 120C for 3 hours was no longer seen in biomass after 14 days of compos ting. Even when total bi odegradation was not yet achieved, PLA had apparently disappeared, and it could be said that breakdown was complete. 83

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However, continued production of CO2 attributed to PLA material suggests the presence of PLA, probably as oligomers, and lactic acid. It was also observed that more severe tr eatments (i.e. 120C x 3h) did not create a lag period for adaptation or conditioning In these samples, the rate of biodegradation was very high at the beginning of the experiment and then decreased over time. In contrast, PLA samples less severely treated (i.e. 120C x 1h) showed a sigmoidal behavior, represented by a lag period, accelerating and decel erating stage. It is said that a material must fulfill three conditions to be compostable [49]: (a) it must biodegrade quickly, (b) it must not a lter the quality of the compost, and (c) it must disintegrate. Samples of steam-treated PLA fulfilled these conditions. The 31-days biomass, consisting of biodegraded treated PLA in compost, had sim ilar appearance, texture and odor as compost without PLA. The pH of the final biomass (~6.6-6.8) did not change (Table 6-1). Pictures of the compost with and without treated PLA are shown in Figure 6-5. There was no apparent difference between the two compost samples. Kinetics of Steam-Treated PLA Biodegradation in Compost Nonlinear regression to fit e xperimental data to the logi stic model was obtained using SigmaPlot v.10. Outputs are shown in Table 6-2. Parameters of the model are related with th e pattern and magnitudes of the biodegradation curves. The parameter a is the plateau, which is the maximum value of biodegradation that can be achieved. In all cases the value of a is close to 100 which is the theoretical plateau. The parameter b is associated with the lag period, so smalle r values indicate shorter times, and larger values of b indicate longer times to start biodegrading at a high rate. This matches experimental results, where more severe pretreatments resulted in lower values of b (for instance, steamtreated PLA at 120C x 3h got the shortest value of b, and untreated PLA the highest). Finally, 84

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the parameter xo is the time at which half of the biodegradation will be completed. Thus, larger values of xo indicate longer total tim es for biodegradation. This explains why untreated PLA biodegradation curve had a much higher values of this parameter. The goodness of fit was excellent in all curves as represented by r-square values very close to unity. Figure 6-6 shows experimental data and predic ted values from the model. Weight Loss of Steam-Treated PLA in Compost and Comparison with other Common Feedstock Figure 6-7 shows results of weight loss of steam-treated PLA (120C x 3h, 4h), wood and virgin corrugated paperboard in 6-month mature compost. Treated PLA samples were the only ones that broke apart inside the compost. Screen ed envelopes were designed to retain broken parts for further weighting. After 14 days, steam -treated PLA achieved weight losses of 94.9% (120C x 4h) and 86.4% (120C x 3h), whereas w ood and corrugated board achieved values of 0.9% and 39.2%, respectively. These results dem onstrate that PLA subjected to steam (120C x 3 and 4 h) breaks down much faster than wood and virgin corrugated paperboard, which are usually accepted in composting facilities. Figur es 6-8, 6-9 and 6-10 show pictures of these samples, and it was observed that steam-tr eated PLA was most greatly affected. Conclusion It has been demonstrated that steam-treated PL A is affected very significantly in compost, breaking down even faster than common organi c feedstock universally accepted in composting facilities such as wood and virgin corrugated paperboard. Polylactic acid treated with steam at 120C for 3, 2 and 1 hours, achieved degr adability (60% of conversion to CO2) after 14, 16 and 19 days, whereas untreated PLA did not achie ve biodegradability even after 31 days. Degradability was evidenced by complete PLA disappearance. Additiona lly, resulting compost did not appear to be affected by a loadi ng of about 10% by weight PLA in compost. 85

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Characteristics of the final compost when steam-t reated PLA was initially present were similar than those of the compost by itself. The thr ee requirements for compostability are fast breakdown, total disintegration and no alteration; thus, steam-treated PLA should be considered to be compostable. Biodegradation kinetics of PLA fit very well us ing the proposed logistic model with three parameters, and provides valuable informati on for understanding biodegradation behavior. Determined parameters confirmed that pre-comp osting treatments that reduced PLA molecular weight provided head start and acceleration effects during subsequent composting process. Parameter b from the model is associated with the lag period and therefore, with the head start. The acceleration effect is associated with the derivative of the model after the lag period. Figure 6-1. Main reactions in PLA biodegradation 86

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Figure 6-2. Head start effect Figure 6-3. Acceleration effect Slope 1 Slope 2 % biod. % biod. Time 87

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0 20 40 60 80 100 0 5 1015 2025 3035Days% Biod. Untreated 120C x 1h 120C x 2h 120C x 3h Figure 6-4. Biodegradation of steam-t reated PLA over time in compost Figure 6-5. Biodegraded PLA in compost: (a) com post by itself, (b) compost + PLA (not seen any more) 88

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0 20 40 60 80 100 0 5 101520253035Days% biodegradation Untreated 120C x 1h 120C x 2h 120C x 3h Figure 6-6. Logistic model fit for PLA biodegradation data 94.9 86.4 39.2 0.90 10 20 30 40 50 60 70 80 90 100 PLA 4h @ 120CPLA 3h @ 120CCorr PB WoodMaterialWeight Loss (%) Figure 6-7. Weight loss of steam-treated PLA in compost compared with corrugated board and wood. 89

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Figure 6-8. Corrugated paperboard s ubjected to compost for 14 days Figure 6-9. Wood subjected to compost for 14 days Figure 6-10. Steam treated PLA (120C x 3h) subjected to compost for 14 days 90

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Table 6-1. pH of biomass (compost + biodegraded PLA) Sample pH Compost 0h @ 120C 1h @ 120C 2h @ 120C 3h @ 120C 6.7 6.6 6.6 6.6 6.8 Table 6-2. Parameters of the logi stic model (%biod = a / (1 + (t/xo)-b) Untreated 1h @ 120C 2h @ 120C 3h @ 120C a b xo R-square 84.01 4.103 26.45 0.9819 75.15 3.612 12.57 0.9981 96.91 2.04 12.61 0.9981 113.8 1.05 12.49 0.9981 91

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CHAPTER 7 CONCLUSIONS Among all the treatments intended to reduce PL A molecular weight, exposure to steam is the most effective, achieving a reduction of 94% when subjecte d to steam at 120C for 4 hours. The main mechanism for this event is therma l hydrolysis. Gamma irra diation is also a good treatment for reducing PLA molecular weight, ac hieving a reduction of 86% when absorbed dose was 172 kGy. Steam treatment of PLA is a process that hyd rolyzes the polymer molecule until complete de-polymerization to lactic acid units, as it wa s confirmed by FTIR-ATR analysis. At 120C for 24h, PLA weight loss was 84.7% sugges ting significant conversion to la ctic acid. It is believed that steam treatments may be suitable not onl y for making PLA waste more accommodating to commercial composting operations, but also for assi sting with separation of PLA from traditional plastic wastes. The model that describes molecula r weight reduction in PLA as a consequence of hydrolytic steam treatment follows firs t order kinetics with activation energy, Ea, of 52.3 KJ/mol. To evaluate PLA biodegradati on in compost, the most reli able and user friendly method designed and developed was the method of perf orated jars. This method does not require a complex and expensive gas supply system as the one proposed in the standards (ASTM D5338 and ISO 14855) and is much easier to conduct and adapt. Under anaerobic conditions, untreated PLA did not biodegrade at mesophilic temperature. Polylactic acid did degrade under thermophilic conditions. Treated PLA, using steam or irradiation, degraded in mesophilic or thermophili c conditions, and at higher rates than untreated PLA. For anaerobic digestion, steam-treatment was more effective than gamma irradiation treatment, and gamma irradiation treatment was more effective than electron-beam irradiation. The best results observed were for the most se vere treatment studied, which was steam at 120C 92

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for 3 hours, followed by anaerobic digestion under thermophilic conditions, where methane yield was 225 ccCH4/g after 56 days. Under aerobic conditions it was confirmed that steam-treatment provi ded head start and acceleration effects to PLA biodegradation. Ground PLA subjected to steam at 120C for 3 hours achieved biodegradability (60% of conversion as CO2) in 14 days, whereas untreated PLA did not achieve this state even after 31 days. It was verified that steam-treated PLA (120C x 3h) undergoes breakdown even faster than virg in corrugated paper board and wood, common feedstock universally acc epted in composting plants. In addi tion, at a rate of 10% of compost feedstock, steam-treated PLA does not appear to alter the compost during the time it is breaking down. Three findings of this work are significant. First, it was found that PLA biodegrades under anaerobic thermophilic conditions. This finding is important because post-consumer PLA material may be used in anaerobic digestion fo r energy recovery, instead of being treated as waste disposal. Second, a simple and reliable method to determine biodegradation of polymers under composting conditions was developed. This method, named method of perforated jars, may be a valuable contribution to biodegradation assessments and may serve as a more convenient and less expensive a lternative to current standard methods. Finally, steam-exposure can be also seen as a potential technique for recycling PLA. This is possible since, under steam conditions, PLA hydrolyzes to lac tic acid which is soluble in wa ter, and therefore, may be separated from the plastic waste stream and re-polymerized into virgin PLA. There are big differences between biodegradable, bioerodable and compostable. PLA is slightly biodegradable and bioerodable in compos ting conditions. However, this is insufficient for commercial composting operations. Steam tr eatments may help to separate PLA from 93

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municipal solid waste streams as well as accelerating conversion of the material. Such treatments may play an important role in helping to keep PLA waste out of sanitary landfills. The ability to easily compost PLA and/or other biopolymers help s to complete the cycle that is the very essence of sustainability. 94

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CHAPTER 8 RECOMMENDATIONS FOR FUTURE WORK Main recommendations for future work are: Evaluate steam-treated PLA biodegradati on in real composting conditions where temperature varies. Evaluate lactic acid recovery from a plas tic waste downstream containing PLA material, by using steam treatment. Find selective microbial strains capable to optimize PLA biodegradation under aerobic, anaerobic and facultative conditions. Investigate other ways to recove r lactic acid from PLA, such as treating dissolved alkalitreated PLA. 95

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APPENDIX A CHARACTERISTICS OF THE COMPOST The compost was originally developed using a standard organic matter feedstock recipe developed in this study. It consisted of freshly cut grass from the University of Floridas golf course (58%), saw dust (11%), virgin corrugate d board (11%) and mature compost (20%). All raw materials were prepared and mixed toge ther in a rotational composter (CompostTwin, Mantis, Southampton PA) placed in a com puter controlled environmental chamber (Environmental Growth Chambers, Chagrin Falls Ohio) at 35C .This formulation met the requirement of initial optimum nutrient balance ca rbon/nitrogen ratio of 30. Periodic addition of water provided appropriate mois ture content between 55% and 70% to promote and maintain biological activity. Initial net weight in the com poster was 118 lb and ro tation was applied daily to ensure adequate mixing and aeration. Main fu tures of the 3 months old mature compost are shown in Table A-1. Table A-1. Characteristics of prepared mature compost Features Value Method Moisture Ash Volatile solids Size pH CO2 generation rate 47-53 % 22-27 % 21-24 % 4.6 mm, max 6.8 0.14 moles/day/Kg(*) Weight of dry matter/ initial weigh (105C) Weight of residue after 550 for 6 hours VS = 100 %Moisture %Ash Manual screening pH-meter, dilution 1:5 Flow method, variable depending on age (*) Estimated using the flow method at 3 months old. 96

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APPENDIX B PERMEABILITY TO CARBON DIOXIDE To determine the permeability of the plastic film to CO2, the cells of the oxygen transmission rate analyzer Oxtran 2/20 ST (Mocon, Inc., Minneapolis MN) were modified in order to connect to the carbon dioxide se nsor of the headspace analyzer Pac Check 650 (Mocon, Inc., Minneapolis MN). Carbon dioxide was used in place of oxygen, and the carrier gas was N2/H2 (96/4). A sketch of the system is shown in Figure B-1. Permeability to CO2 at 15, 25 and 35C was estimated using Equation B-1 after carbon dioxide concentration, [CO2], measured by the sensor, became constant (steady state). The thickness of the film, E, was m easured with a micrometer and was 3.035 mil with an area of 100 cm2. The universal gas constant is R = 82.057 atm-cc/molK. The gas flow was set at 20cc/min (at 21.1C, 1atm) using needle valves of the Oxtran 2/20 ST. ATR E]CO[Flow P2 gas CO2 (B-1) Permeability to CO2 at 58C was estimated using Arrhenius equation shown in Equation B-2, with Ea = 23.08 KJ/mol and pre-expone ntial factor Po =5 8614 mol-mil/atm-d-m2. The final value obtained was PCO2 @ 58C = 13.41 mol-mil/atm-d-m2. )RT/Eexp(PPa o (B-2) Parameters of the Arrhenius equation, Ea and Po, were determined from linearization of experimental data of permeability at 15, 25 and 35C. The slope of the plot ln PCO2 vs 1/T is equal to Ea/R, and the intercept is ln Po (Figure B-2). The gas constant R for the Arrhenius equation was R = 0.0083 KJ/K-mol. 97

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Physical cells (Oxtran 2/20 ST) N2H2 CO2 Film Figure B-1. Mounted system to determine CO2 transmission rate 1/T (K-1) 0.003200.003250.003300.003350.003400.003450.00350 ln P 1.2 1.4 1.6 1.8 2.0 2.2 Figure B-2. Arrhenius plot for activation energy determination (CO2 permeability) CO2 + N2H2 N2H2 + CO2 CO2 sensor ( Pac Check 650 ) Flow-meter Ea = 23.08 KJ/mol Po = 58614 mol.mil/(atm.d.m2) 98

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APPENDIX C VOLUME OF BIOMASS Volume of biomass inside a container was de termined from weight of biomass and its density (Equation C-1). biomass containerinbiomassW V (C-1) To determine density, a biomass sample was inserted and compacted into a vessel of known weight and volume. Then the filled vessel was weighed and the density was calculated by the ratio of net weight over vol ume, as shown in Equation C-2. vesselV )emptyvessel(W)fullvessel(W (C-2) The vessel used in these an alyses was a bottle cap with weight = 5.8728 g and capacity (volume) = 6.3 cc. 99

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APPENDIX D BAG VOLUME Bag volume refers to the inte rnal volume occupied by head space gases and biomass. Bag volume, Vbag, was determined by total volume, Vbag tot, minus the volume of the film itself, Vfilm (Equation D-1). Vbag = Vbag tot Vfilm (D-1) Total volume, Vbag tot, was measured using a volume-meter designed and built for this study, and uses the principle of water displacem ent. Figure D-1 describes functionality. Water warmed to experimental conditions (e.g. 58C) was used as the disp lacement fluid. Errors due to depth pressure were predicte d to be minimal (< 0.1%). Volume of the film was calculated by mu ltiplying length by width by thickness of the opened bag. This was about Vfilm = (12+12)(9)(3.035 x 0.00254) = 1.67 cm3. Figure D-1. Volume-meter designed for bag volume determination. 100

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APPENDIX E OVERALL COEFFICIENT OF DIFFUSION Jars (cap.936cc) with same f eatures as those used for the method of perforated jars (biodegradation assessment) were filled with 20 ml of DI water and some carbon dioxide. Jars were closed using the 5-hole perforat ed lids and stored in a Lab-Line L-C Incubator (Lab-Line Instruments, Inc., Melrose Park, IL) at 58 C for 5.5 hours. Each 0.5 h, the headspace was analyzed for CO2 concentration and the rate of effusi on was estimated. Results are shown in Table E-1. The percentage of CO2 adjusted was obtained from the empirical exponential model that best-fitted experimental data. Only those values where %CO2 was below 5.7% were taken into account for the regression since they better re presented values in normal assessments. The parameters of the model were obtained in MS Excel, with r-square of 0.998 (Figure E-1). Volume of CO2 adjusted in the headspace expressed in cubic centimeters, is the product of the CO2 concentration adjusted by the volume of the headspace (936 20 = 916cc). Then, the number of moles was determined by the Idea l Gases Law (moles = PV/RT) assuming that expansion of water due to temperature was negligib le. Finally, the rate of effusion was calculated by Equation E-1. time hsinmoles rate Effusion (E-1) Effusion rate was plotted against %CO2 in the headspace (Figure 4-12) and the following relationship was found (Equation E-2); )CO(%00031.0rate Effusion2 (E-2) Equation E-2 shows that the overall co efficient of diffusion is 0.00031 moles CO2/h per percentage unit. 101

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Table E-1. Carbon dioxide effusion rate estimation through 5-hole lid Time %CO2 %CO2 cc CO2 moles Effusion rate moles CO2/h (h) exp. adj. adj. CO2 in hs released 0.0 28.1 38.8 355.3 0.01308 1.0 15.9 18.3 167.7 0.00617 0.00691 1.5 11.2 12.6 115.2 0.00424 0.00386 2.0 7.8 8.6 79.1 0.00291 0.00265 2.5 5.7 5.9 54.3 0.00200 0.00182 3.0 4.1 4.1 37.3 0.00137 0.00125 3.5 2.9 2.8 25.6 0.00094 0.00086 4.0 1.9 1.9 17.6 0.00065 0.00059 4.5 1.4 1.3 12.1 0.00045 0.00041 5.0 0.9 0.9 8.3 0.00031 0.00028 5.5 0.6 0.6 5.7 0.00021 0.00019 Figure E-1. Carbon dioxide concen tration in headspace over time 102

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APPENDIX F NUTRIENT FORMULA FO R ANAEROBIC MEDIA Table F-1. Anaerobic media formulation Chemical Concentration (g/L) Amount Resazurin (NH4).2HPO4 Ca Cl2.2H2O H3BO3 H2WO4 FeCl2.4H2O Na2S.9H2O Biotin Folic acid Vitamin B12 Sodium bicarbonate DI water 1 26.7 16.7 0.38 0.007 370 500 0.002 0.002 0.0001 1.80 ml 5.40 ml 27.00 ml 2.70 ml 0.27 ml 18.00 ml 18.00 ml 1.80 ml 0.90 ml 0.18 ml 8.40 g Up to 2 L 103

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BIOGRAPHICAL SKETCH I was born in Lima, Peru, in 1970 where I lived almost all my life. I graduated as Food Engineer (1992) and MS in Food Technology (1997) both at Universidad Nacional Agraria la Molina in my home country. In 1995, I married and became a professor at the Food Engineering Department in my former university. I spent 10 years of my life teaching, training, managing and researching in the area of food engineering. In 2005, I was awarde d a fellowship granted by the Organization of the American Stat es (OAS) to pursue the Ph.D. progr am at University of Florida through the Agricultural and Biol ogical Engineering Department / Packaging Science Program. I have spent my last three years studying topics related to pack aging and doing research in the field of biodegradable pack aging, under Dr. Bruce Welt. 108