ANAEROBIC DIGESTION OF LDPE/LLDPE BLEND FILM AND PET SHEET WITH PRODEGRADING ADDITIVES AT 35 AND 50°C
By
Tuan Anh Nguyen

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Packaging – Master of Science
2014

ABSTRACT
ANAEROBIC DIGESTION OF LDPE/LLDPE BLEND FILM AND PET SHEET WITH PRODEGRADING ADDITIVES AT 35 AND 50°C
By
Tuan Anh Nguyen
Low density polyethylene/linear low density polyethylene blend film and polyethylene
terephthalate sheet incorporated with pro-degrading additives from Symphony Environmental
Ltd., Wells Plastics Ltd., and EcoLogic LLC were evaluated in an anaerobic digestion
environment for 16 months together with negative (blank) and positive controls (cellulose) in
general accordance with ASTM

D5526-12. Total biogas production of cellulose was

significantly higher than that of the remaining samples. Total biogas production of samples
containing plastics and the negative control were not significantly different from each other. Prodegrading additives tested in the study did not increase the biodegradation of these plastic
materials.

To my family

iii

ACKNOWLEDGEMENTS

I would like to express my deep gratitude to Dr. Susan Selke, Dr. Rafael Auras, and Dr.
Yan Liu for their insight and guidance. I would like to acknowledge the Vietnam Education
Foundation and the Center for Packaging Innovation and Sustainability at the School of
Packaging, Michigan State University (MSU) for their financial support.
I would like to express my sincere appreciation to Dr. Phu Nguyen, Department of
Forestry for his continuous encouragement during my undergraduate and graduate years at MSU.
In addition, I would like to thank Rijosh Cheruvathur, Edgar Castro, Tanatorn Tongsumrith, Rui
Chen, Zhenhua Ruan, Yuan Zhong, Hayati Samsudin, Siyuan Tian, Ning Gong, Alyssa Petz,
Matt Weal from the School of Packaging and the Department of Biosystems and Agricultural
Engineering, and those who I unintentionally did not mention.

iv

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................................ vii
LIST OF FIGURES ........................................................................................................................ x
KEY TO ABBREVIATIONS ....................................................................................................... xii
CHAPTER 1: INTRODUCTION ................................................................................................... 1
1.1
Background ..................................................................................................................... 1
1.1.1
Functions of packaging ............................................................................................. 1
1.1.2
Low density polyethylene/linear low density polyethylene and polyethylene
terephthalate ............................................................................................................................ 1
1.1.3
Legislation and public opinions in the United States and around the world on
plastic waste ............................................................................................................................ 2
1.1.4
Common biodegradable plastics and pro-degrading additives in the market ........... 3
1.1.5
Skepticism of biodegradable technology .................................................................. 4
1.2
Motivation ....................................................................................................................... 5
1.3
Goal and objectives ......................................................................................................... 6
CHAPTER 2: LITERATURE REVIEW ........................................................................................ 7
2.1
LDPE, LLDPE, their production and general properties ................................................ 7
2.2
PET, its production and general properties ..................................................................... 9
2.3
The concept of polymer biodegradation ....................................................................... 10
2.4
Factors affecting biodegradation of polymers .............................................................. 10
2.5
History of biodegradable polymers and common approaches in making polymers
biodegradable ............................................................................................................................ 12
2.6
Oxo-biodegradation mechanism ................................................................................... 13
2.7
Pro-degrading additives used in the project and their mechanisms .............................. 14
2.8
Publicly known/evidence about biodegradation with these or other additive systems . 15
2.9
Anaerobic digestion: mechanism, inhibitors, and other influencing factors ................ 15
2.10 Testing standards .......................................................................................................... 17
2.11 ASTM D5526-12 .......................................................................................................... 19
CHAPTER 3: MATERIALS AND METHODS .......................................................................... 20
3.1
Introduction ................................................................................................................... 20
3.2
Plastics manufacturing and properties .......................................................................... 20
3.3
Anaerobic digestion inoculum and dairy manure ......................................................... 23
3.4
Sample preparation ....................................................................................................... 24
3.5
Biogas measurement ..................................................................................................... 25
3.6
Optical microscopy ....................................................................................................... 26
3.7
Spiking of the bioreactors ............................................................................................. 27
3.8
pH determination .......................................................................................................... 27
v

3.9

Statistical analysis and data management ..................................................................... 28

CHAPTER 4: RESULTS AND DISCUSSION............................................................................ 29
4.1
Total gas evolution ........................................................................................................ 29
4.2
Spiking .......................................................................................................................... 35
4.3
Optical microscopy ....................................................................................................... 39
CHAPTER 5: CONCLUSIONS ................................................................................................... 42
5.1
Overall conclusions ....................................................................................................... 42
5.2
Recommendations for future work ............................................................................... 42
APPENDICES .............................................................................................................................. 44
APPENDIX A: WEIGHT OF SAMPLES ................................................................................ 45
APPENDIX B: ACCUMULATED GAS MEASUREMENT .................................................. 49
APPENDIX C: SPIKING GAS MEASUREMENT DATA ..................................................... 81
APPENDIX D: ANOVA TABLES .......................................................................................... 82
APPENDIX E: BOXPLOTS..................................................................................................... 83
APPENDIX F: MATLAB CODE FOR PLOTTING MAIN EXPERIMENT DATA ............. 87
APPENDIX G: MATLAB CODE FOR PLOTTING SPIKING EXPERIMENT DATA ....... 89
APPENDIX H: MATLAB CODE FOR STATISTICAL ANALYSIS .................................... 91
APPENDIX I: MAXIMUM THEORETICAL GAS EVOLUTION FORMULA ................... 94
APPENDIX J: WEIGHT OF MANURE .................................................................................. 95
APPENDIX K: AUTOCAD DRAWINGS OF GAS MEASURING APPARATUS .............. 96
APPENDIX L: GAS MEASUREMENT FOR ORIGINAL POSITIVE CONTROLS ............ 97
BIBLIOGRAPHY ......................................................................................................................... 99

vi

LIST OF TABLES

Table 2-1. A comparison of blown film properties between LDPE and LLDPE, adapted from
[44]. ................................................................................................................................................. 8
Table 2-2. Overview of testing standards for biodegradation of plastic materials under anaerobic
conditions. ..................................................................................................................................... 18
Table 3-1. Average thickness of the LDPE/LLDPE film and PET sheet produced. .................... 22
Note: LDPE/LLDPE 0 wt% thickness was 0.9 ± 0.2 mil, and PET 0 wt% thickness was 9.2 ± 0.6
mil. ................................................................................................................................................ 22
Table 3-2. Carbon, nitrogen, and hydrogen content for samples. ................................................. 22
Table 3-3. Composition for treatments and controls. ................................................................... 24
Table 4-1. Average accumulated gas volume at day 252 (for manure 2nd run and cellulose
samples) and 464 (for manure 1st run and LDPE/LLDPE samples) at 35°C. ............................... 30
Table 4-2. Average accumulated gas volume at day 252 (for manure 2nd run and cellulose
samples) and 464 (for manure 1st run and LDPE/LLDPE samples) at 50°C. ............................... 30
Table 4-3. Average accumulated gas volume at day 252 (for manure 2nd run and cellulose
samples) and 464 (for manure 1st run and PET samples) at 35°C. ............................................... 31
Table 4-4. Average accumulated gas volume at day 252 (for manure 2nd run and cellulose
samples) and 464 (for manure 1st run and PET samples) at 50°C. ............................................... 31
Table A-1. Weight for LDPE/LLDPE samples (35 °C). .............................................................. 45
Table A-2. Weight for PET samples (35 °C). ............................................................................... 46
Table A-3. Weight for cellulose samples (35 °C). ........................................................................ 46
Table A-4. Weight for LDPE/LLDPE samples (50 °C). .............................................................. 47
Table A-5. Weight for PET samples (50 °C). ............................................................................... 48
Table A-6. Weight for cellulose samples (50 °C). ........................................................................ 48
Table B-1. Accumulated gas measurement for LDPE/LLDPE 0 wt% (35 °C). ........................... 49
Table B-2. Accumulated gas measurement for LDPE/LLDPE Ecologic 1 wt% (35 °C). ........... 50

vii

Table B-3. Accumulated gas measurement for LDPE/LLDPE Ecologic 5 wt% (35 °C). ............ 51
Table B-4. Accumulated gas measurement for LDPE/LLDPE Symphony 1 wt% (35 °C). ......... 52
Table B-5. Accumulated gas measurement for LDPE/LLDPE Symphony 5 wt% (35 °C). ......... 53
Table B-6. Accumulated gas measurement for LDPE/LLDPE Wells 1 wt% (35 °C). ................. 54
Table B-7. Accumulated gas measurement for LDPE/LLDPE Wells 5 wt% (35 °C). ................. 55
Table B-8. Accumulated gas measurement for PET 0 wt% (35 °C). ........................................... 56
Table B-9. Accumulated gas measurement for PET Ecologic 1 wt% (35 °C). ............................ 57
Table B-10. Accumulated gas measurement for PET Ecologic 5 wt% (35 °C). .......................... 58
Table B-11. Accumulated gas measurement for PET Wells 1 wt% (35 °C). ............................... 59
Table B-12. Accumulated gas measurement for PET Wells 5 wt% (35 °C). ............................... 60
Table B-13. Accumulated gas measurement for manure only (1st run) (35 °C). .......................... 61
Table B-14. Accumulated gas measurement for manure only (2nd run) (35 °C). ......................... 62
Table B-15. Accumulated gas measurement for cellulose 0.55g (35 °C). .................................... 63
Table B-16. Accumulated gas measurement for cellulose 1.10g (35 °C). .................................... 64
Table B-17. Accumulated gas measurement for LDPE/LLDPE 0 wt% (50 °C). ......................... 65
Table B-18. Accumulated gas measurement for LDPE/LLDPE Ecologic 1 wt% (50 °C). .......... 66
Table B-19. Accumulated gas measurement for LDPE/LLDPE Ecologic 5 wt% (50 °C). .......... 67
Table B-20. Accumulated gas measurement for LDPE/LLDPE Symphony 1 wt% (50 °C). ....... 68
Table B-21. Accumulated gas measurement for LDPE/LLDPE Symphony 5 wt% (50 °C). ....... 69
Table B-22. Accumulated gas measurement for LDPE/LLDPE Wells 1 wt% (50 °C). ............... 70
Table B-23. Accumulated gas measurement for LDPE/LLDPE Wells 5 wt% (50 °C). ............... 71
Table B-24. Accumulated gas measurement for PET 0 wt% (50 °C). ......................................... 72
Table B-25. Accumulated gas measurement for PET Ecologic 1 wt% (50 °C). .......................... 73

viii

Table B-26. Accumulated gas measurement for PET Ecologic 5 wt% (50 °C). .......................... 74
Table B-27. Accumulated gas measurement for PET Wells 1 wt% (50 °C). ............................... 75
Table B-28. Accumulated gas measurement for PET Wells 5 wt% (50 °C). ............................... 76
Table B-29. Accumulated gas measurement for manure only (1st run) wt% (50 °C)................... 77
Table B-30. Accumulated gas measurement for manure only (2nd run) wt% (50 °C). ................. 78
Table B-31. Accumulated gas measurement for cellulose 0.55g (50 °C). .................................... 79
Table B-32. Accumulated gas measurement for cellulose 1.10g (50 °C). .................................... 80
Table C-1. Spiking gas measurement for bioreactors at 35 °C. .................................................... 81
Table C-2. Spiking gas measurement for bioreactors at 50 °C. .................................................... 81
Table D-1. ANOVA table for LDPE/LLDPE samples and controls at 35 °C. ............................. 82
Table D-2. ANOVA table for PET samples and controls at 35 °C. ............................................. 82
Table D-3. ANOVA table for LDPE/LLDPE samples and controls at 50 °C. ............................. 82
Table D-4. ANOVA table for PET samples and controls at 50°C. .............................................. 82
Table J-1. Manure mixture for main experiment. ......................................................................... 95
Table L-1. Accumulated gas measurement for starch (original positive control) (35 °C). .......... 97
Table L-2. Accumulated gas measurement for cellulose (original positive control) (35 °C). ...... 97
Table L-3. Accumulated gas measurement for starch (original positive control) (50 °C). .......... 97
Table L-4. Accumulated gas measurement for cellulose (original positive control) (50 °C). ...... 98

ix

LIST OF FIGURES

Figure 2-1. Differences in structures of LDPE, LLDPE, and single-site-catalyzed LLDPE,
adapted from [45]............................................................................................................................ 8
Figure 2-2. Factors affecting polymer biodegradation, adapted from [50]................................... 11
Figure 2-3. Mechanism of free-radical oxidation of polyethylene, adapted from [55]. ............... 14
Figure 2-4. Anaerobic digestion process, adapted from [59]....................................................... 16
Figure 3-1. Gas production measuring apparatus. ........................................................................ 26
Figure 3-2. Data structure diagram of a cell array. ....................................................................... 28
Figure 4-1. Accumulated gas in mL at 35°C for LDPE/LLDPE Ecologic 1 & 5 wt%,
LDPE/LLDPE Symphony 1 & 5 wt%, LDPE/LLDPE Wells 1 and 5 wt%, cellulose 0.55g and
1.10 g (positive controls), and blanks (manure 1st and 2nd run). ................................................... 32
Figure 4-2. Accumulated gas in mL at 50°C for LDPE/LLDPE Ecologic 1 & 5 wt%,
LDPE/LLDPE Symphony 1 & 5 wt%, LDPE/LLDPE Wells 1 and 5 wt%, cellulose 0.55g and
1.10 g (positive controls), and blanks (manure 1st and 2nd run). ................................................. 33
Figure 4-3. Accumulated gas in mL at 35°C for PET Ecologic 1 & 5 wt%, PET Wells 1 & 5
wt%, cellulose 0.55g and 1.10 g (positive control), and blanks (manure 1st and 2nd run). ......... 34
Figure 4-4 Accumulated gas in mL at 50°C for PET Ecologic 1 & 5 wt%, PET Wells 1 & 5 wt%,
cellulose 0.55g and 1.10 g (positive control), and blanks (manure 1st and 2nd run). .................. 35
Figure 4-5. Spikes in accumulated gas evolution at 35°C for LDPE/LLDPE 0 wt%,
LDPE/LLDPE Ecologic 5 wt%, LDPE/LLDPE Symphony 5 wt%, LDPE/LLDPE Wells 5 wt%,
and blank (bioreactor 1, 9, 14, 21, and 44). .................................................................................. 36
Figure 4-6. Spikes in accumulated gas evolution at 50°C for LDPE/LLDPE 0 wt%,
LDPE/LLDPE Ecologic 5 wt%, LDPE/LLDPE Symphony 5 wt%, LDPE/LLDPE Wells 5 wt%,
and blank (bioreactor 53, 60, 64, 71, and 94). .............................................................................. 37
Figure 4-7. Spikes in accumulated gas evolution at 35°C for PET Ecologic 5 wt%, PET Wells 5
wt%, and blank (bioreactor 28, 36, and 44). ................................................................................. 38
Figure 4-8. Spikes in accumulated gas evolution at 35°C for PET Ecologic 5 wt%, PET Wells 5
wt%, and blank (bioreactor 73, 81, 85, and 94). ........................................................................... 39
Figure 4-9. Optical microscopy of the surface of a LDPE/LLDPE Symphony 5 wt% sample from
bioreactor #13 (10x objective, 100x total). ................................................................................... 40
x

Figure 4-10. Optical microscopy of the surface of a LDPE/LLDPE Symphony 5 wt% sample
from bioreactor #13 (100x objective, 1000x total). ...................................................................... 40
Figure 4-11. Optical microscopy of the surface of a PET Wells 5 wt% sample from bioreactor
#34 (10x objective, 100x total). .................................................................................................... 41
Figure 4-12. Optical microscopy of the surface of a PET Wells 5 wt% sample from bioreactor
#34 (100x objective, 1000x total). ................................................................................................ 41
Figure E-1. Boxplots for LDPE/LLDPE samples and controls at 35 °C. ..................................... 83
Figure E-2. Boxplots for PET samples and controls at 35 °C. ..................................................... 84
Figure E-3. Boxplots for LDPE/LLDPE samples and controls at 50 °C. ..................................... 85
Figure E-4. Boxplots for PET samples and controls at 50 °C. ..................................................... 86
Figure K-1. Dimensions of the components of the gas measuring apparatus. .............................. 96

xi

KEY TO ABBREVIATIONS

C

=

carbon

C/N

=

carbon-nitrogen ratio

CH4

=

methane

CO2

=

carbon dioxide

d

=

days

E

=

Ecologic

g

=

gram

H2O

=

water

LDPE

=

low density polyethylene

LLDPE

=

linear low density polyethylene

mL

=

milliliters

mm

=

millimeters

NaOH

=

sodium hydroxide

O2

=

oxygen

PET

=

polyethylene terephthalate

S

=

Symphony

UV

=

ultraviolet light/radiation

W

=

Wells

wt%

=

Weight percent

xii

CHAPTER 1: INTRODUCTION

1.1

Background

1.1.1

Functions of packaging
The three functions of packaging are protection, utility, and communication [1]. For food

and beverage packaging, protection is probably the most important because of its direct effects
on products’ shelf-lives. Packaging helps prevent spoilage due to environmental, chemical, and
physical hazards associated with the production, transportation, and distribution of food and
beverages. In 2010, food and beverage packaging accounted for 69% of the global market for
consumer packaging. Categorizing by materials, plastic topped all other packaging materials
with 37% of the global consumer packaging market by value [2]. Because of its protective
function, plastic packaging is generally inert to biological and chemical changes, and continues
to exist in the environment hundreds to thousands of years past its useful life [3]. This creates
severe problems for waste management around the world.

1.1.2

Low density polyethylene/linear low density polyethylene and polyethylene

terephthalate
Two common packaging plastics for food and beverages are low density
polyethylene/linear low density polyethylene (LDPE/LLDPE) and polyethylene terephthalate
(PET). LDPE/LLDPE is widely used in plastics bags, film for bakery goods, shrink films,
overwrap, pallet stretch wrap, and milk/juice cartons [4]. PET food applications include
containers for carbonated beverages, water, and juice [5]. Since their introduction in the
twentieth century, packaging plastics’ production, consumption, and waste generation has

1

increased significantly [6]. Since 1980, the amount of PET from bottles and jars generated in the
U.S. municipal solid waste (MSW) has increased tenfold from 260 to 2,670 thousand tons. In
2010, LDPE/LLDPE from containers and packaging generated in the U.S. MSW accounted to
3,480 thousand tons, with 12.1% recovery. In the same year, PET from containers and packaging
generated in MSW totaled 3,380 thousand tons, only 23.1% of which was recovered [7].

1.1.3

Legislation and public opinions in the United States and around the world on plastic

waste
In 2012, Barnosky et al. reported in Nature that the Earth’s ecological system is
“approaching a planetary-scale critical transition as a result of human influence”. Similar to
localized ecosystem shifts that are suddenly and irreversible, the Earth’s state shift will have
detrimental effects on our lives [8]. These concerns about the impacts of people on the Earth
have focused more attention on the issue of disposal of plastics. Legislation has been introduced
around the world to deal with the plastic waste problem, although the approach has not been
systematic. The best known legislation is the ban of the plastic bag, the most ubiquitous of all
packaging. In Bangladesh, the plastic bag ban started around the capital city of Dhaka in 2002
and quickly spread nationwide. Shoppers were encouraged to use alternatives such as jute, paper,
and reusable cloth bags [9]. In 2002, Ireland began to tax plastic shopping bags at a rate of €0.15
initially and increased to €0.22 per bag. Bag use was down 90% shortly after the ban, with strong
support from the public and the retail industry [10]. In 2007, San Francisco became the first city
in the US to ban plastic checkout bags in large supermarkets and retail pharmacies. On
September 2012, the ordinance was upheld by the San Francisco Superior Court banning “noncompostable plastic checkout bags [in] all retail stores and food establishments, and imposing a

2

10-cent charge on other bags provided to consumers” [11]. Since 2010, shoppers in Washington,
D.C. buying food or alcohol must pay a $0.05 bag fee for each plastic bag used [12]. In
Australia, a bag ban took effect in the state of South Australia in May 2009, the Northern
Territory in September 2011, and the Australian Capital Territory in November 2011. Since the
ban in each state or territory, retailers are only allowed to provide compostable or biodegradable
bags that meet Australia’s standard to customers [13–15]. In 2012, The United Arab Emirates
(UAE) banned all disposable plastic bags with the exception of those made from oxobiodegradable plastic, in compliance with UAE Standard 5009:2009 [16].

1.1.4

Common biodegradable plastics and pro-degrading additives in the market
To cope with changes in legislation and consumer perception, two prominent trends that

have emerged in plastics manufacturing are producing biodegradable plastics from biomass
sources (biodegradable bioplastics) and adding degradation-promoting additives to petroleumbased plastics. With the advance of technology, bioplastics’ properties and processability are
improving but still somewhat inferior to those of traditional petroleum-based plastics. Some
examples of commercial biodegradable packaging materials based on raw materials from crops
are Mater-Bi, NatureWorks Polylactide, Bioska, Bioplast, Solanyl, Potatopac, Greenfil and EcoFoam [17]. Because of the drawbacks in processability of biodegradable bioplastics,
degradation-promoting additives are being marketed as the better option [18]. Many degradationpromoting additives are oxo-biodegradable additives, most often stearates incorporated with
3+,

transition metal ions such as Fe

2+

Mn , or Co

2+

[19]. Some examples of degradation-

promoting additives on the market include Totally Degradable Plastic Additives [20], VIBATAN
04089 [21], d2w [22], Eco-One [23], Reverte [24], and EcoPure [25].
3

1.1.5

Skepticism of biodegradable technology
In the UAE, Wells Plastics Reverte and Symphony’s d2w were certified to be in

compliance with UAE Standard 5009:2009 [26,27]. With the ban of plastic bags in the UAE in
2012, only bags made from plastics incorporated with Wells Plastics’ and Symphony’s or other
approved suppliers’ additives are allowed to circulate in the country. Around the world, retailers
such as U.S.’s Yoke’s, United Kingdom’s Co-operative Food, and Vietnam’s Saigon Co-op
supermarket chains also picked up oxo-biodegradable plastic bags [28–30].
However, there have been a variety of criticisms about the oxo-biodegradable
technology. In a report to the United Kingdom’s Department for Environment, Food and Rural
Affairs, researchers from Loughborough University (Leicestershire, UK) highlighted consumers’
confusion about oxo-biodegradable claims, the inability of oxo-biodegradable plastics to be
composted, and their effects on recycling and composting facilities [31]. As the result of this
finding, the UK’s Co-operative Food supermarket chain decided to stop using oxo-biodegradable
plastic bags [32]. Some plastic trade associations also expressed concerns and doubts about the
oxo-biodegradable technology. The Flexible Packaging Association and the Society of the
Plastics Industry Bioplastics Council published positions on degradation-promoting additives
that asked manufacturers to include scientific data from recognized third parties to corroborate
claims such as “biodegrades in landfills” or “oxo-biodegradable”. Claims must be tested
according to accepted industry standards such as ASTM D6400, ASTM D6868, ASTM D7081
or EN 13432 [33,34]. In addition, degradation-promoting additives do not have the support of
recyclers because of the belief that common plastics incorporated with biodegradable additives
can contaminate recyclers’ processing operations. In its 2010 strategy paper, the European
Plastics Recyclers Association calls bioplastics and oxo-biodegradables unsustainable. They also

4

asked for collection of these materials to be in a separate stream because of the fear that
bioplastics and oxo-biodegradable plastics will have damaging effects on mechanical recycling
[35]. In addition, the Association of Postconsumer Plastic Recyclers shared the same concerns
about the largely unknown effects of oxo-biodegradable plastics on recycled materials [36].
In October 2012, the Federal Trade Commission (FTC) amended its Guides for the Use
of Environmental Marketing Claims. The guide was originally published in 1992, amended in
1996 and 1998 subsequently. The guide advises manufacturers and marketers to possess data to
qualify their environmental marketing claims. Without these data, manufacturers can be found by
the FTC to deceive consumers which can result in orders prohibiting their deceptive marketing as
well as fines [37,38]. FTC has been regularly taking actions against companies for deceptive
environmental claims. Examples of companies that have received fines from the FTC include
Amazon.com Inc., Leon Max Inc., Sears, Roebuck and Co., Kmart Corporation, Tender, and
Dyna-E [39,40].

1.2

Motivation
There is great interest among environmentally responsible companies in using

degradation-promoting additives for plastic packaging. LDPE/LLDPE film was chosen because
Bimbo Bakeries USA, one of the project’s sponsors, was interested in the additives’ application
in bread bags. Bimbo Bakeries is the largest bakery company in the US whose brands include
Arnold, Bimbo, Boboli, Sara Lee, Thomas’, Oroweat, and many others [41]. In addition to
LDPE/LLDPE film, PET sheet was chosen as PET is widely used for carbonated soft drinks,
water, ketchup, and many other beverages and food. This is of particular interest of member
companies of the Center for Packaging Innovation and Sustainability (CPIS), Michigan State

5

University (East Lansing, MI USA). CPIS, the main sponsor of this project, “is a global leader in
research and outreach related to packaging innovation and sustainable systems, resulting in
positive environmental effects on the global footprint of packaging and related systems across
the supply chain” [42]. CPIS’s members are The Coca-Cola Company, ConAgra Foods, The
Dow Chemical Company, Abbott Laboratories, World Wildlife Fund, H. J. Heinz Company, and
AkzoNobel [43].
In 2010, with 12.1% and 23.1% recovery for LDPE/LLDPE and PET from containers and
packaging in U.S. MSW, a significant amount was discarded. Most of these plastics were
discarded to landfills or combusted [7]. Therefore, it is the project’s interest to study the
biodegradation of LDPE/LLDPE and PET with pro-degrading additives in landfill conditions.

1.3

Goal and objectives
The goal of this study was to investigate the performance of degradation-promoting

additive systems from Symphony Environmental Ltd., Wells Plastics Ltd., and EcoLogic LLC in
an anaerobic digestion environment for 16 months. The objective was to study the
biodegradation of LDPE/LLDPE and PET in an anaerobic digestion environment conditions by
measuring total biogas production.

6

CHAPTER 2: LITERATURE REVIEW

2.1

LDPE, LLDPE, their production and general properties
LDPE was discovered by the Imperial Chemical Industries in 1933. It is produced by the

free-radical-initiated polymerization process from ethylene monomers. Ethylene monomers are
mainly manufactured by manufactured from natural gas or high temperature cracking of crude
oil. As a result of the free-radical polymerization process, LDPE has a large amount of longchain branching. The molecular weight, molecular weight distribution, frequency of short-chain
branches, and frequency and length of the long-chain branches of LDPE affect its physical and
extrusion properties [44].
LLDPE was introduced for commercial use in the late 1970s by Union Carbide and Dow
Chemical. LLDPE is produced by the copolymerization of ethylene and α-olefins. As a result,
LLDPE has a narrower molecular weight distribution than LDPE and does not contain longchain branching. Because of its nature as a copolymer, LLDPE’s properties are strongly
dependent on comonomer content. The four most common comonomers are 1-hexene (40%), 1butene (35%), 1-octene (25%), and 4-methyl-1-pentene (only a small fraction) [45]. The
difference in structure of LDPE, LLDPE, and single-site-catalyzed LLDPE is illustrated in figure
2-1. A comparison of blown film properties between LDPE and LLDPE is shown in table 2-1.

7

LLDPE

HP LDPE

mLLDPE
Figure 2-1. Differences in structures of LDPE, LLDPE, and single-site-catalyzed LLDPE,
adapted from [45].

Table 2-1. A comparison of blown film properties between LDPE and LLDPE, adapted from
[44].
Property
Melt index, g/10 min
Density, g/cm3
Comonomer
Dart drop, N/mm
Puncture energy, kJ/m
Elmendorf tear, N/mm
(=dyn/cm)
MD
XD
Tensile strength, MPa
MD
XD
Haze, %
Gloss, 45°

ASTM test
method
D1238
D1505
D1709

HPLDPE
2.5
0.921
None
29
27

HPLDPE
0.2
0.923
None
71
22

62
43
20
19
6
70

LLDPE

LLDPE

LLDPE

1.0
0.918
Butene
39
71

1.0
0.918
Hexene
77
76

1.0
0.918
Octene
97
-

35
39

54
131

131
226

143
309

19
21
25
30

35
26
17
53

36
32
20
50

45
35
12
60

D1922

D882

D1003
D2457

8

2.2

PET, its production and general properties
High molecular weight PET was first successfully synthesized in England in 1942 by J.

Rex Whinfield and W. Dickson. However, commercialization of PET did not commence until
after World War II ended. At first, PET was manufactured to be used exclusively as synthetic
fibers. PET was not widely used as a molding resin in cold molds, with temperatures less than
130 °C, due to its low crystallization rate. However, during the late 1960s, specific nucleating
agents, whose development was spearheaded by Akzo and DuPont, removed this technical
disadvantage. PET is made by the reaction of ethylene glycol and terephthalic acid or dimethyl
terephthalate. Terephthalic acid is produced by air-oxidizing p-xylene in acetic acid under
moderate pressure with the help of catalysts. Since the 1967-1972 period, direct esterification
using pure terephthalic acid has been favored over the dimethyl terephthalate method due to the
improved polymerization and purification processes of terephthalic acid. However, in recent
years, due to an increase in recycling of PET, the dimethyl terephthalate method has had a
resurgence. Dimethyl terephthalate can be made from the methanolysis and glycolysis of waste
PET [46].
The degree of crystallinity of PET dictates its thermochemical properties. The usual
melting temperature is from 260-265 °C but can reach 280 °C for highly annealed samples. PET
is semipermeable to oxygen and carbon dioxide. The stretch blow molding process used to create
PET bottles involves radial and axial drawing, which causes strain-induced crystallization. This
crystallization improves the mechanical strength and reduces the permeability of the bottles [46].

9

2.3

The concept of polymer biodegradation
According to ASTM, a “biodegradable plastic is a degradable plastic in which the

degradation results from the action of naturally-occurring micro-organisms such as bacteria,
fungi, and algae” [47]. Biodegradation of polymers is typically a surface erosion process. The
long chains and water-insolubility of polymers make them unsuitable for being transported
directly into the microorganisms to be digested. The process starts with the secretion of
extracellular enzymes by the microorganisms. The products of this stage are then transported into
the microorganisms to be digested. End products include water, carbon dioxide, methane, and
new biomass [48]. The materials must not have negative impacts on either the disposal processes
or the environment [49].

2.4

Factors affecting biodegradation of polymers
Biodegradation is affected by the exposure conditions and the characteristics of the

polymer. Figure 2-2 illustrates the relationship. Exposure conditions can be classified as abiotic
and biotic. Abiotic factors include but are not limited to temperature, pH, moisture, and UV
exposure. Microbial activity tends to increase at higher temperature and moisture content.
However, extremely high temperature can slow down and stop the microbial activity. pH level
can affect hydrolysis of polymers. In addition, pH values below or above the range that
microorganisms can tolerate can slow down or stop the microbial activity. Another factor, UV
exposure, affects biodegradation by causing main chain scissions (hastens up biodegradation)
and introducing crosslinking (slows down biodegradation) [50]. Biotic factors include but are not
limited to extracellular enzymes, hydrophobicity, and biosurfactants. Extracellular enzymes are
used by the microorganisms to depolymerize the polymer outside the cell wall [48,50].

10

Temperature
Moisture
Abiotic

Factors affecting polymer biodegradation

pH
UV radiation

Exposure
Conditions

Extracellular
enzymes
Biotic

Hydrophobicity

Flexibility

Biosurfactants

Crystallinity
Morphology
Functional Groups
Crosslinking
Polymer
Characteristics
Molecular Weight
Copolymers
Blend
Tacticity
Additives

Figure 2-2. Factors affecting polymer biodegradation, adapted from [50].

11

Polymer biodegradation is also dictated by the characteristics of the polymer itself. These
characteristics include but are not limited to flexibility, crystallinity, morphology, functional
groups, crosslinking, molecular weight, copolymers, blends, tacticity, and additives. A rise in
conformational flexibility of a polymer increases the accessibility of microorganisms and water
to the polymer. On the other hand, crystallinity can affect the biodegradation greatly by affecting
the accessibility of water. The more crystalline the polymer is, the more difficult it is for water to
diffuse through the polymer. For this same reason, adding a copolymer causes molecular
irregularity, which in turn decreases the crystallinity and increases biodegradation. However, it
should be noted that addition of copolymer can increase the rigidity of the polymer and reduce its
biodegradability. Hydrolysable functional groups act as sites for hydrolysis for many polymers.
Crosslinking reduces the accessibility of microorganisms and water to the polymer chains. Since
only low molecular weight polymer molecules can be transported into the cell wall for digestion,
high molecular weight polymers take longer to biodegrade [50].

2.5

History of biodegradable polymers and common approaches in making polymers

biodegradable
Because polyethylene is hydrophobic, usually incorporated with antioxidants and
stabilizers during processing, and has high molecular weight but no functional groups, it is not
considered a biodegradable polymer [51]. Many attempts have been made since the 1970s to
achieve biodegradable polyethylene. One of the approaches is to use polyethylene-starch blends.
Starch, is a relatively cheap commodity which primarily comes from cereal crops. However,
even though starch comes from renewable sources, there are concerns about the sustainability of
using starch for plastics manufacturing and the conflict with food production. Starch-based

12

blends are more expensive and there is currently not adequate infrastructure for recycling and
composting polyethylene-starch blends [52]. A different approach is to incorporate oxobiodegradable additives into polyethylene. In addition to initiating the free-radical oxidation of
polyethylene, these additives can modify the surface of polymer to be hydrophilic [53]. Common
oxo-biodegradable additives contain transition metal stearates. Transition metals commonly used
are manganese (Mn2+/Mn3+), iron (Fe2+/Fe3+), and cobalt (Co2+/Co3) [54].

2.6

Oxo-biodegradation mechanism
Figure 2-3 illustrate the free-radical oxidation process of polyethylene. The cycle starts

from the top of the diagram with the creation of free radical P. from PH due to shear stress or
catalyst residues. The free radical P. reacts with oxygen to form POO.. POO. then reacts with a
polymer molecule to form a new radical P. and POOH. The pro-degrading additive catalyzes the
conversion of POOH to PO. and .OH. PO. will be converted to biodegradable functional
fragments Fs(O)x. .OH will react with a new polymer molecule PH to form POH that will
eventually be converted by further oxidation and fragmentation. In summary, in one cycle, the
radical will react with two polymer molecules PHs to form new radicals. These new radicals will
travel in the same process all over again.

13

PH
Shear stress/
Catalyst residues

O2

POH + H2O

PH

P.

POO.

Further oxidation
& fragmentation

PH
PO. + .OH

Fs(O)x
Biodegradable
functional fragments

∆/hv
Pro-degrading
additive

POOH

Figure 2-3. Mechanism of free-radical oxidation of polyethylene, adapted from [55].

2.7

Pro-degrading additives used in the project and their mechanisms
d2w additive from Symphony is claimed to work according to an oxo-biodegradation

mechanism [22]. On the other hand, Reverte from Wells plastics uses a hybrid mechanism. In the
first stage, the company claims that the additive catalyzes the oxo-biodegradation of the polymer.
In the second stage, the microbial growth is said to be promoted by the additive [24]. The
mechanism for Eco-One additives from Ecologic is not explained in detail. The company asserts
that the additives promote the formation of biofilm on the surface of the plastics, and expand the
molecular structure of the plastic so that microorganisms can penetrate and digest the plastics
[23].

14

2.8

Publicly known/evidence about biodegradation with these or other additive systems
Corti et al. reported that combining abiotic treatment such as prolonged thermal and

sunlight exposure, and oxo-biodegradable additive promotes the biodegradation of LLDPE films
containing oxo-biodegrdable additives inoculated with fungal strains known for their ability to
use oxidized LDPE as the only carbon source. Films with oxo-biodegradable additives also have
higher carbonyl indices and produce more CO2 in fungal biodegradation tests [51]. Billingham et
al. reported that LDPE incorporated with EPI’s TDPA oxo-biodegradable additive degraded
rapidly in thermal aging. In experiments monitored by FTIR spectroscopy, tensile testing, and
size exclusion chromatography, films with oxo-biodegradable additives lose strength and
polymer chain length quickly, and produce oxidation products [56]. Chiellini et al. studied the
effects of temperature and relative humidity on oxidation and cleavage of the macromolecules by
measuring weight variation (using an analytical balance), film wettability (by measuring contact
angle on glass slides), carbonyl index (using FTIR), molecular weight (using size exclusion
chromatography), and extractability with polar solvents of oxidized thermal-aged samples using
EPI’s TDPA additive. They concluded that the TDPA oxo-biodegradable additive was effective
in initiating the oxidative degradation of the polymer [57]. Vogt and Kleppe showed that after
exposure to light, polyethylene and polypropylene with 2% Renatura pro-oxidant additive
continued to degrade under dark thermal conditions. The thermal oxidative degradation increases
with the increase in light exposure [58].

2.9

Anaerobic digestion: mechanism, inhibitors, and other influencing factors
Anaerobic digestion is a complicated biological process. As illustrated in figure 2-4, there

are multiple reactions running in series and parallel to each other. There are four main stages.

15

The first stage is the hydrolysis of complex organic materials such as proteins, carbohydrates,
and lipids into amino acids, carbohydrates, fatty acids, and alcohols. The second stage is the
fermentation of amino acids and carbohydrates. The fermentation of amino acids produces shortchain fatty acids, succinate, aminovalerate, and H2. The fermentation of soluble carbohydrates
results in ethanol, acetate, H2, and CO2.The third stage is the anaerobic oxidation of long-chain
fatty acids and alcohols. The end products are acetate and propionate. The fourth stage is the
anaerobic oxidation of short-chain fatty acids such as propionate and butyrate to acetate and
H2.The last stage is methanogenesis. The end products are CH4 and CO2 [59].

COMPLEX POLYMERS
HYDROLYSIS
FATTY ACIDS, ALCOHOLS

AMINO ACIDS, SUGARS
FERMENTATION

INTERMEDIARY PRODUCTS
(Propionate, Butyrate, etc.)

ACETATE
HOMOACETOGENESIS
ACETICLASTIC
METHANOGENESIS

METHANE
CARBON DIOXIDE

Figure 2-4. Anaerobic digestion process, adapted from [59].

16

ANAEROBIC
OXIDATION

HYDROGEN
CARBON DIOXIDE
REDUCTIVE
METHANOGENESIS

There are many factors that can inhibit an anaerobic digestion process. Ammonia exists
in large quantity in animal waste due to decomposition of organic nitrogen. The mechanisms for
ammonia inhibition include intracellular pH changes, increase in maintenance energy
requirements, and specific enzyme reaction inhibition. Ammonia inhibition affects methanogens
the most. Light metal ions such as Na, K, Mg, Ca, and Al also inhibit the anaerobic digestion
process. A high concentration of salt causes the cells of the bacteria to lose water due to osmotic
pressure. Light metal ions come from the breakdown of biomass or are added to adjust pH. At
low or moderate levels, these micronutrients can speed up bacterial growth. However, at high
levels, light metal ions can inhibit or even halt the anaerobic digestion process. Organic
chemicals can also inhibit an anaerobic digestion process. Agricultural waste contains high
amounts of lignocellulosic content in stalks, straws, and bark. Methanogens are highly
vulnerable to lignin and lignin derivatives [60]. Temperature can also affect the kinetics of the
anaerobic digestion process. Higher temperature (below or equal to the optimum temperature)
leads to higher microbial activity. However, temperature beyond the optimum temperature
decreases microbial activity [59].

2.10

Testing standards

Testing standards for biodegradation of plastic materials under anaerobic conditions include:
•

ASTM D5526-12: Standard Test Method to Determine Anaerobic Biodegradation of
Plastic Materials under Accelerated Landfill Conditions.

•

ASTM D5511-12: Standard Test Method for Determining Anaerobic Biodegradation of
Plastic Materials under High-Solids Anaerobic-Digestion Conditions.

17

•

ASTM D5210-92(2007): Standard Test Method for Determining the Anaerobic
Biodegradation of Plastic Materials in the Presence of Municipal Sewage Sludge.

•

ASTM D7475-11: Standard Test Method for Determining the Aerobic Degradation and
Anaerobic Biodegradation of Plastic Materials under Accelerated Bioreactor Landfill
Conditions.

Table 2-2. Overview of testing standards for biodegradation of plastic materials under anaerobic
conditions.
Test standards

Purpose

Data obtained

Anaerobic Biodegradation
Test duration, % biodegradation as a
ASTM

of Plastic Materials Under
function of time, % CH4 and % CO2 in

D5526-12

Accelerated Landfill
evolved gas [61].
Conditions
Anaerobic Biodegradation
Test duration, % biodegradation as a

ASTM

of Plastic Materials Under
function of time, % CH4 and % CO2 in

D5511-12

High-Solids Anaerobicevolved gas [62].
Digestion Conditions
Test duration, % of gas evolution as a
Anaerobic Biodegradation

ASTM

function of time, molecular weight of
of Plastic Materials in the

D5210-

plastic before and after the exposure,
Presence of Municipal

92(2007)

weight loss of the specimen, inoculum’s
Sewage Sludge
soluble solid organic carbon content [63].

18

Table 2-2. (cont’d)
Temperature range of the test as a
Aerobic Degradation and
function of time, test duration, %
Anaerobic Biodegradation of
biodegradation as a function of time, %
ASTM D7475-11

Plastic Materials under
CH4 and % CO2 in headspace, changes in
Accelerated Bioreactor
molecular weight, weight, tensile, and
Landfill Conditions
other properties of the samples [64].

2.11

ASTM D5526-12
The ASTM D5526-12 test method simulates biologically active landfills where moisture

and temperature are controlled, and gas recovery is promoted. There are seven steps in the
method. The first step is to choose and evaluate the test material. The second step is to obtain a
pretreated municipal-solid-waste medium and an anaerobic inoculum. The third step is to place
the material in an anaerobic static batch fermentation. It is noted that the medium should contain
more than 30% solids. The fourth step is to quantify the total carbon (in CO2 and CH4 evolved)
as a function of time. The fifth step is to clean and test the exposed material. The sixth step is to
calculate the degree of biodegradability. The last step is to assess the degree of biodegradability
when the conditions are less than optimum [61].

19

CHAPTER 3: MATERIALS AND METHODS

3.1

Introduction
This chapter describes the production of materials, and methodology employed to study

the biodegradation of LDPE/LLDPE film and PET sheet in an anaerobic digestion environment.
The major challenge of this research was to provide enough nutrients for the anaerobic
microorganisms to thrive while keeping the closed system’s pH and environmental parameters at
optimal growth conditions.

3.2

Plastics manufacturing and properties
The polymeric film and sheet used in this study were extruded at the School of

Packaging, Michigan State University (East Lansing, MI). The resins for the LDPE/LLDPE film,
DOWLEX 2045G (LLDPE) and DOW 501I (LDPE), were donated by the Dow Chemical
Company (Midland, MI). LDPE and LLDPE resins were blended in a 70/30 ratio by weight.
LDPE/LLDPE blend was then mixed with 1 and 5 wt% of degradation promoting masterbatch
additives d2w (Symphony Environmental Ltd., Borehamwood, Hertfordshire, UK), Reverte
(Wells Plastics Ltd., Stone, Staffordshire, UK), and Eco-one EL10 (EcoLogic LLC, Oakbrook
Terrace, IL). The film was extruded on a Killion KLB 100 blown film extruder (Davis-Standard
LLC, Pawcatuck, CT) with a screw diameter of 25.4 mm (2 inch), screw length/ diameter ratio of
24:1, and a 2 in diameter circular die. The temperature profile of the extruder was 215-215-212212-210-204 ºC (420-420-415-415-411-410-400 ºF) for barrel zones 1, 2, 3, clamp ring, adapter,
die 1, and die 2, respectively. A screw speed of 14 rpm and take up speed of 10 feet per minute
were used. The diameter of the film was controlled at 10 cm at a blow up ratio of 2. The overall

20

thickness of the LDPE/LLDPE control film was 0.9 ± 0.2 mil. Table 3-1 shows the overall
thickness of the produced film.
PET resin, provided by EcoLogic LLC (Oakbrook Terrace, IL), was mixed with 1 and 5
wt% of degradation promoting masterbatch additives Reverte (Wells Plastics Ltd., Stone,
Staffordshire, UK) and Eco-one EC 80 (EcoLogic LLC, Oakbrook Terrace, IL). The resin was
placed in a vacuum oven at 110 °C for 24 hours for drying. After drying, the resin was stored
under vacuum and was cooled down to room temperature. Resin was removed from storage just
before extrusion to prevent any regain of moisture. PET sheet was manufactured by cast film
extrusion using a Microextruder model RCP-0625 (Randcastle Extrusion Systems, Inc., Cedar
Grove, NJ). The microextruder has a 1.5875 cm (0.625 inch) diameter 24/1 L/D ratio extruder
with 34 cc volume. The extrusion system was equipped with a 20 cm (8 in) wide coat hanger die,
Eurotherm temperature control system for the extruder (Eurotherm, Ashburn, Virginia), a chill
roll with Sterling M50-3-2-2 cooling system (Sterling, New Berlin, WI) and a Bronco II take up
roll from Seco AC/DC drives (Warner Electric, Braintree, MA). The temperature profile of the
extruder was 218-226-257-254-254 ºC (425-500-495-490-490 ºF) for feed zone, barrel zones 2,
3, transfer tube, and die, respectively. A screw and take up speed of 60 rpm were used. The chill
roll temperature was controlled at 71 ºC (160 ºF) and was set at a speed of 15 rpm. The chill roll
was placed very close to the die exit so that the film was quenched rapidly in order to prevent
crystallization of the film, resulting in a highly amorphous film. Table 3-1 shows the overall
thickness of the produced sheet.

21

Table 3-1. Average thickness of the LDPE/LLDPE film and PET sheet produced.

LDPE/LLDPE
PET

Percent loading of additive,
%
1
5
1
5

Ecologic,
mil
1.0 ± 0.3
1.1 ± 0.3
11.2 ± 1.1
12.4 ± 0.8

Wells Plastics,
mil
1.0 ± 0.2
1.1 ± 0.1
9.4 ± 0.5
9.0 ± 0.3

Symphony,
mil
1.3 ± 0.3
1.0 ± 0.2
N/A
N/A

Note: LDPE/LLDPE 0 wt% thickness was 0.9 ± 0.2 mil, and PET 0 wt% thickness was 9.2 ± 0.6
mil.
The total amount of carbon, nitrogen and hydrogen content for each sample was
determined by a CHN analyzer from Perkin Elmer (Waltham, Massachusetts). Values are
provided in Table 3-2.

Table 3-2. Carbon, nitrogen, and hydrogen content for samples.
Sample Name
LDPE Control
LDPE Ecologic 1 wt%
LDPE Ecologic 5 wt%
LDPE Wells 1 wt%
LDPE Wells 5 wt%
LDPE Symphony 1 wt%
LDPE Symphony 5 wt%
PET Control
PET Ecologic 1 wt%
PET Ecologic 5 wt%
PET Wells 1 wt%
PET Wells 5 wt%

C, wt%
84.8 ± 1.4
85.2 ± 0.5
84.6 ± 0.4
85.6 ± 0.5
85.5 ± 0.4
85.5 ± 0.4
85.2 ± 0.6
62.1 ± 0.6
61.9 ± 0.8
61.8 ± 0.9
61.7 ± 0.9
61.5 ± 1.2

22

H, wt%
14.5 ± 0.5
14.4 ± 1.0
14.7 ± 0.2
14.7 ± 0.6
15.1 ± 0.2
15.1 ± 0.2
14.9 ± 0.3
4.2 ± 0.1
4.2 ± 0.0
4.2 ± 0.1
4.1 ± 0.2
4.0 ± 0.2

N, wt%
0.1 ± 0.1
0.1 ± 0.0
0.1 ± 0.0
0.1 ± 0.0
0.1 ± 0.1
0.1 ± 0.0
0.1 ± 0.1
0.1 ± 0.0
0.1 ± 0.1
0.1 ± 0.1
0.1 ± 0.1
0.1 ± 0.1

3.3

Anaerobic digestion inoculum and dairy manure
This experiment was done in general accordance with ASTM D5526-12. This test

method simulates biologically active landfills where moisture and temperature are controlled,
and gas recovery is promoted. The standard was used as a starting point. There were deviations
that will be discussed here. Even though the standard only covers tests at 35 ± 2°C, the tests were
run at 35 and 50 °C. In addition, pretreated-household waste was replaced by dairy manure.
Manure was used because of its high biological activity. The standard requires that the total solid
content be more than 30%. However, a total solid content of 5% was used because lower solid
content could lead to higher yield [65]. The standard suggested cellulose (analytical grade for
thin-layer chromatography) as a positive control. In the test, we used a powder form of cellulose
as well as corn starch.
LDPE/LLDPE film and PET sheet produced were exposed to anaerobic digestion
environments at 35 and 50 °C. The anaerobic inoculum was obtained directly from an
operational in-house anaerobic digester at Michigan State University. In this digester, pretreated
household waste was replaced by fresh dairy manure as permitted by ASTM D5526-12. The
manure was obtained from the Michigan State University dairy farm and added with water to
create a 5% (w/v) total solids mixture. The weights of the manure used for the mixtures are
included in Appendix J. Manure was used as the only nitrogen source. Carbon sources were
manure and plastics. Manure also acts as a buffer to maintain the pH within the optimal range
(6.8 – 7.2) so that microorganisms can grow and thrive. The treatments and controls were
mounted on orbital shakers model Innova 2050 (New Brunswick Scientific, Edison, New Jersey)
at 95 ± 5 RPM, and placed in incubators model 11-690 (Fisher Scientific, Hampton, New
Hampshire) at 35 and 50°C.

23

3.4

Sample preparation
LDPE/LLDPE film and PET sheet with or without additives were cut into 0.635 cm x

0.635 cm (0.25 in x 0.25 in) pieces using a sample cutter and scissors. They were then weighed
before being inserted into 125 mL serum bioreactors. The weight of each sample is listed in
Appendix A. These serum bioreactors were airtight and fitted with septa for measuring gas
production. The weight of each component of the mixture was calculated to yield a C/N ratio
within the optimum 20 – 30 range. Table 3-3 shows the composition of each treatment and
control.

Table 3-3. Composition for treatments and controls.

Negative control (blank)
Positive control 1 (cellulose)
Positive control 2 (cellulose)
Treatment (LDPE/LLDPE)
Treatment (PET)

Inoculum,
mL
7.5
7.5
7.5
7.5
7.5

Manure,
mL
75
75
75
75
75

Cellulose or
starch, g

Plastic
sample, g

0.550
1.100
2.250
3.085

Initially, positive controls containing 4.337 g of starch or cellulose were digested rapidly
resulting in uncontrollable drop in pH below 5 (Appendix L contains gas evolution data for these
bioreactors). This created an unfavorable living environment for microorganisms inside the
bioreactors. Therefore, a new experiment with just the negative control (blank) and two positive
controls was conducted adding 0.55 and 1.10 g of cellulose for the positive controls using the
same manure and inoculum. The theoretical total gas evolution of a bioreactor containing manure
is calculated to be 1.21 L and 1.27 L at 35°C and 50°C, respectively. The theoretical total gas
evolution of a bioreactor containing 0.55g cellulose is calculated to be 1.57 L and 1.65 L at 35°C
24

and 50°C, respectively. The theoretical total gas evolution of a bioreactor containing 1.10g
cellulose is calculated to be 1.93 L and 2.03 L at 35°C and 50°C, respectively. The formula used
for the theoretical values is included in Appendix I.

3.5

Biogas measurement
The generation of total gas in mL (i.e., methane, carbon dioxide and other minor gases)

from the LDPE/LLDPE and PET samples without and with additives was quantified, and
compared to both positive and negative controls. The gas production was measured using the
water displacement method (as depicted in Figure 3-1) initially every 3 days (for the first 100
days) and then after every 7 days. A glass water reservoir with a capacity of 1000 mL was filled
with 800 mL of water. Two metal tubes were inserted through a rubber stopper attached to the
opening of the water reservoir. One tube was fitted securely inside Tygon tubing, also connected
to a needle on the other end. To measure the gas, the needle was inserted into the septum on top
of the bioreactors, and the tube connected to the Tygon was placed inside a graduated cylinder to
collect the displaced amount of water. The system was entirely airtight except the openings
where biogas entered and water siphoned out. The excess pressure inside the bioreactor pushed
the water level inside the water reservoir down and siphoned water out of the tubing. The
measurement was terminated when the whole system returned to atmospheric pressure. The
needle was then removed from the septum of the bioreactor. The AutoCAD drawings of the
bioreactor and the gas measuring apparatus are included in Appendix K.
Bioreactors were taken out of the chamber in batches of three in order to keep the inner
temperature of the bioreactor close to the original temperature as much as possible. Each

25

measurement took 20 seconds to finish. The water was refilled when the water level reached the
40% mark. Gas measurements are in Appendix B and C.

Figure 3-1. Gas production measuring apparatus.

3.6

Optical microscopy
After the main experiment is finished, plastic samples from bioreactors 2, 13, 23, 34, 52,

59, 70, and 75 were retrieved. Biofilms on the surface of the plastic samples were examined
under a compound microscope model Eclipse 50i (Nikon Instruments Inc., Melville, NY) with

26

10 x and 100 x objective for 100 x and 100 x total system magnification. Images were captured
with Nikon’s NIS-Elements D 3.00.

3.7

Spiking of the bioreactors
After 464 days of running the initial experiment, 0.55 g of corn starch was added to one

replicate of LDPE/LLDPE 0 wt%, LDPE/LLDPE Ecologic 5 wt%, LDPE/LLDPE Symphony 5
wt%, LDPE/LLDPE Wells 5 wt%, PET Ecologic 5 wt%, PET Wells 5 wt%, and blank at each
incubation temperature. The biogas production as well as the pH level was monitored for the
following 50 days.

3.8

pH determination
The pH of each bioreactor was checked several times during the study to ensure that it

was close to 6.9. A controlled environment anaerobic chamber model 855 from Plas Labs, Inc.
(Lansing, MI) was used to conduct this determination. The headspace gas (85% nitrogen, 10%
hydrogen, and 5% carbon dioxide) was supplied by Airgas Inc. (Radnor Township,
Pennsylvania). The bioreactors were shaken before being opened. A pH meter model Accumet
AB15 (Fisher Scientific) with an Ag/AgCl electrode was inserted into the opening. If the pH was
lower than 6.7, NaOH 10% solution was added to bring it close to a pH of 6.9.
+

At low concentration, Na can stimulate anaerobic bacteria growth. However, at higher
+

concentrations, Na slows down and even inhibits bacteria growth by disrupting their
+

+

metabolisms [60]. The half maximal inhibitory concentration of Na , the amount of Na needed

27

to inhibit the growth of anaerobic bacteria by half, is 5.6 to 53 g/L [65]. In the experiment, the
total amount of NaOH added to each bioreactor was less than 4 g/L.

3.9

Statistical analysis and data management
Statistical analyses were performed using MATLAB (The MathWorks Inc., Natick, MA).

One-way analyses of variance (ANOVA) were performed, and Tukey’s honestly significant
difference (HSD) test was used to determine differences (p≤0.05) among treatments and controls
at day 252 (for manure 2nd run and cellulose samples) and 464 (for manure 1st run and the rest).
The MATLAB code for ANOVA analysis is included in Appendix H. Gas measurement data
were recorded in Excel files, transferred into MATLAB, and saved as MAT-files (“LDPE
35C.mat”, “LDPE 50C.mat”, “PET 35C.mat”, “PET 50C.mat”, “LDPE 35C spike.mat”, “LDPE
50C spike.mat”, “PET 35C spike.mat”, and “PET 50C spike.mat”). Each MAT-file contains a
cell array named “data” or “spikedata”. In each cell array, there are two columns. The first cell
array’s column contains the name of the samples. The corresponding rows on the second column
contain matrices. Each matrix contains two columns (day and corresponding total accumulative
gas).

Figure 3-2. Data structure diagram of a cell array.
28

CHAPTER 4: RESULTS AND DISCUSSION

4.1

Total gas evolution
One-way analyses of variance (ANOVA) were performed, and Tukey’s HSD test was

used to determine differences (p ≤ 0.05) among treatments and controls at day 252 (for manure
2nd run and cellulose samples) and 464 (for manure 1st run and the rest). Appendix E contains
boxplots accompanying the ANOVA operations. Appendix D contains ANOVA tables listing
sum of squares, mean squares, degree of freedom for treatment and errors, f-ratios, and p-values.
Because all p-values (LDPE/LLDPE 35°C vs. controls, LDPE/LLDPE 50°C vs. controls, PET
35°C vs. controls, and PET 50°C vs. controls) are smaller than α = 0.05, there were significant
differences in total gas evolution among treatments and controls at each temperature. Tukey’s
HSD test was then used for pair-wise comparisons. As shown in Tables 4-1, 4-2, 4-3, 4-4, the
total accumulated gas of cellulose 1.10g and 0.55g were significantly higher compared to blanks
and plastic samples at each temperature. It must be noted that cellulose samples evolved
significantly more biogas in a shorter period of time even though the amounts of carbon in the
cellulose samples were less than a quarter of those in the plastic samples. On the other hand,
there was no significant difference in gas production between the blanks and the plastic samples.
In addition, at 35 °C, there was no significant difference in gas production between cellulose
1.10g and cellulose 0.55g samples. However, at 50 °C, the gas production of cellulose 1.10g
samples was statistically significantly higher than that of the cellulose 0.55g samples.

29

Table 4-1. Average accumulated gas volume at day 252 (for manure 2nd run and cellulose
samples) and 464 (for manure 1st run and LDPE/LLDPE samples) at 35°C.
Samples
Cellulose 1.10g
Cellulose 0.55g
LDPE/LLDPE Symphony 1 wt%
LDPE/LLDPE Wells 1 wt%
LDPE/LLDPE Ecologic 1 wt%
LDPE/LLDPE Symphony 5 wt%
LDPE/LLDPE Ecologic 5 wt%
LDPE/LLDPE Wells 5 wt%
Manure (1st run)
Manure (2nd run)
LDPE/LLDPE 0 wt%

Mean
1945 a
1818 a
1443 b
1375 b
1373 b
1359 b
1349 b
1319 b
1293 b
1279 b
1266 b

Note: Samples not connected by the same letter are significantly different.

Table 4-2. Average accumulated gas volume at day 252 (for manure 2nd run and cellulose
samples) and 464 (for manure 1st run and LDPE/LLDPE samples) at 50°C.
Samples
Cellulose 1.10g
Cellulose 0.55g
LDPE/LLDPE Wells 1 wt%
Manure (2nd run)
LDPE/LLDPE Wells 5 wt%
LDPE/LLDPE Symphony 5 wt%
LDPE/LLDPE Ecologic 5 wt%
LDPE/LLDPE Symphony 1 wt%
Manure (1st run)
LDPE/LLDPE Ecologic 1 wt%
LDPE/LLDPE 0 wt%

Mean
1973 a
1618 b
1150 c
1088 c
1062 c
1057 c
1024 c
995 c
955 c
952 c
941 c

Note: Samples not connected by the same letter are significantly different.

30

Table 4-3. Average accumulated gas volume at day 252 (for manure 2nd run and cellulose
samples) and 464 (for manure 1st run and PET samples) at 35°C.
Samples
Cellulose 1.10g
Cellulose 0.55g
PET Wells 1 wt%
PET Ecologic 5 wt%
PET Wells 5 wt%
PET 0 wt%
PET Ecologic 1 wt%
Manure (1st run)
Manure (2nd run)

Mean
1945 a
1818 a
1359 b
1348 b
1329 b
1318 b
1296 b
1293 b
1279 b

Note: Samples not connected by the same letter are significantly different.

Table 4-4. Average accumulated gas volume at day 252 (for manure 2nd run and cellulose
samples) and 464 (for manure 1st run and PET samples) at 50°C.
Samples
Cellulose 1.10g
Cellulose 0.55g
PET Wells 1 wt%
Manure (2nd run)
PET Wells 5 wt%
PET 0 wt%
PET Ecologic 5 wt%
PET Ecologic 1 wt%
Manure (1st run)

Mean
1973 a
1618 b
1184 c
1088 c
1048 c
1003 c
997 c
995 c
955 c

Note: Samples not connected by the same letter are significantly different.

Figures 4-1 and 4-2 show the total gas evolution in mL of LDPE/LLDPE samples and
controls at 35 and 50 °C. Figures 4-3 and 4-4 show the total gas evolution in mL of PET samples

31

and controls at 35 and 50 °C. These figures were generated using MATLAB (code in Appendix
F).

Figure 4-1. Accumulated gas in mL at 35°C for LDPE/LLDPE Ecologic 1 & 5 wt%,
LDPE/LLDPE Symphony 1 & 5 wt%, LDPE/LLDPE Wells 1 and 5 wt%, cellulose 0.55g and
1.10 g (positive controls), and blanks (manure 1st and 2nd run).
32

Figure 4-2. Accumulated gas in mL at 50°C for LDPE/LLDPE Ecologic 1 & 5 wt%,
LDPE/LLDPE Symphony 1 & 5 wt%, LDPE/LLDPE Wells 1 and 5 wt%, cellulose 0.55g and
1.10 g (positive controls), and blanks (manure 1st and 2nd run).

33

Figure 4-3. Accumulated gas in mL at 35°C for PET Ecologic 1 & 5 wt%, PET Wells 1 & 5
wt%, cellulose 0.55g and 1.10 g (positive control), and blanks (manure 1st and 2nd run).

34

Figure 4-4 Accumulated gas in mL at 50°C for PET Ecologic 1 & 5 wt%, PET Wells 1 & 5 wt%,
cellulose 0.55g and 1.10 g (positive control), and blanks (manure 1st and 2nd run).

4.2

Spiking
Figures 4-5 and 4-6 showed the spikes in gas production after corn starch was introduced

into bioreactors containing LDPE/LLDPE 0 wt%, LDPE/LLDPE Ecologic 5 wt%,
35

LDPE/LLDPE Symphony 5 wt%, LDPE/LLDPE Wells 5 wt%, PET Ecologic 5 wt%, PET Wells
5 wt%, and blank. The increase in gas production proved that the microorganisms inside the
bioreactor could still grow if enough digestible nutrients were present.

Figure 4-5. Spikes in accumulated gas evolution at 35°C for LDPE/LLDPE 0 wt%,
LDPE/LLDPE Ecologic 5 wt%, LDPE/LLDPE Symphony 5 wt%, LDPE/LLDPE Wells 5 wt%,
and blank (bioreactor 1, 9, 14, 21, and 44).

36

Figure 4-6. Spikes in accumulated gas evolution at 50°C for LDPE/LLDPE 0 wt%,
LDPE/LLDPE Ecologic 5 wt%, LDPE/LLDPE Symphony 5 wt%, LDPE/LLDPE Wells 5 wt%,
and blank (bioreactor 53, 60, 64, 71, and 94).

37

Figure 4-7. Spikes in accumulated gas evolution at 35°C for PET Ecologic 5 wt%, PET Wells 5
wt%, and blank (bioreactor 28, 36, and 44).

38

Figure 4-8. Spikes in accumulated gas evolution at 35°C for PET Ecologic 5 wt%, PET Wells 5
wt%, and blank (bioreactor 73, 81, 85, and 94).

4.3

Optical microscopy
Optical microscopy confirmed with visual inspection that even though biofilms formed

on the surfaces of the plastic samples retrieved, the films were very thin and negligible.

39

Figure 4-9. Optical microscopy of the surface of a LDPE/LLDPE Symphony 5 wt% sample from
bioreactor #13 (10x objective, 100x total).

Figure 4-10. Optical microscopy of the surface of a LDPE/LLDPE Symphony 5 wt% sample
from bioreactor #13 (100x objective, 1000x total).

40

Figure 4-11. Optical microscopy of the surface of a PET Wells 5 wt% sample from bioreactor
#34 (10x objective, 100x total).

Figure 4-12. Optical microscopy of the surface of a PET Wells 5 wt% sample from bioreactor
#34 (100x objective, 1000x total).

41

CHAPTER 5: CONCLUSIONS

5.1

Overall conclusions
The working hypothesis was that the additive systems significantly promote

biodegradation of the polymers into which they are incorporated. This study was run for an
extended amount of time. However, we did not find any evidence of significant degradation of
plastics incorporated with pro-degrading additives. We have concluded that the additive systems
from Symphony Environmental Ltd., Wells Plastics Ltd., and EcoLogic LLC do not promote
significant biodegradation for either LDPE/LLDPE or PET under the anaerobic digestion test
conditions as determined by total gas evolution. This particular study does not prove or disprove
the effects of these additive systems under other test conditions.

5.2

Recommendations for future work
In the future, more research should be done using other additive systems under anaerobic

digestion or aerobic composting conditions. Mechanisms of the biodegradation process can be
studied by constructing mathematical models of the gas production, and comparing their
parameters and corresponding confidence intervals to each other. To construct models that
accurately emulate the response of the system, the concentration of the growth limiting substrate
as well as other critical response variables should be measured. If there is evidence that the
additives promote biodegradation of the plastics, the properties of the degraded samples should
be compared against the properties of samples from the same batch that have not gone through
the degradation process. For example, properties such as intrinsic viscosity, glass transition
temperature, and melting temperature (using differential scanning calorimetry) can be measured.

42

In addition, Fourier transform infrared spectroscopy, UV/Vis spectroscopy, and scanning
electron microscopy can be used to study the chemical structure changes and surface erosion of
the plastics.

43

APPENDICES

44

APPENDIX A: WEIGHT OF SAMPLES

Table A-1. Weight for LDPE/LLDPE samples (35 °C).
Bioreactor

Sample

Weight of plastic sample (g)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21

LDPE/LLDPE 0 wt%
LDPE/LLDPE 0 wt%
LDPE/LLDPE 0 wt%
LDPE/LLDPE Ecologic 1 wt%
LDPE/LLDPE Ecologic 1 wt%
LDPE/LLDPE Ecologic 1 wt%
LDPE/LLDPE Ecologic 5 wt%
LDPE/LLDPE Ecologic 5 wt%
LDPE/LLDPE Ecologic 5 wt%
LDPE/LLDPE Symphony 1 wt%
LDPE/LLDPE Symphony 1 wt%
LDPE/LLDPE Symphony 1 wt%
LDPE/LLDPE Symphony 5 wt%
LDPE/LLDPE Symphony 5 wt%
LDPE/LLDPE Symphony 5 wt%
LDPE/LLDPE Wells 1 wt%
LDPE/LLDPE Wells 1 wt%
LDPE/LLDPE Wells 1 wt%
LDPE/LLDPE Wells 5 wt%
LDPE/LLDPE Wells 5 wt%
LDPE/LLDPE Wells 5 wt%

2.2474
2.2501
2.2413
2.2481
2.2469
2.2434
2.2453
2.2469
2.2539
2.2522
2.2433
2.2527
2.2478
2.2461
2.2477
2.2458
2.2459
2.2494
2.2492
2.2492
2.2476

45

Table A-2. Weight for PET samples (35 °C).
Bioreactor
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36

Sample

Weight of plastic sample (g)
3.0817
3.0840
3.0809
3.0811
3.0838
3.0875
3.0820
3.0847
3.0833
3.0865
3.0812
3.0839
3.0851
3.0864
3.0875

PET 0 wt%
PET 0 wt%
PET 0 wt%
PET Ecologic 1 wt%
PET Ecologic 1 wt%
PET Ecologic 1 wt%
PET Ecologic 5 wt%
PET Ecologic 5 wt%
PET Ecologic 5 wt%
PET Wells 1 wt%
PET Wells 1 wt%
PET Wells 1 wt%
PET Wells 5 wt%
PET Wells 5 wt%
PET Wells 5 wt%

Table A-3. Weight for cellulose samples (35 °C).
Bioreactor
O1
O2
O3
P1
P2
P3

Sample

Weight of sample (g)

Cellulose 0.55g
Cellulose 0.55g
Cellulose 0.55g
Cellulose 1.10g
Cellulose 1.10g
Cellulose 1.10g

0.5540
0.5510
0.5536
1.1029
1.1052
1.1028

46

Table A-4. Weight for LDPE/LLDPE samples (50 °C).
Bioreactor

Sample

Weight of plastic sample (g)

52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72

LDPE/LLDPE 0 wt%
LDPE/LLDPE 0 wt%
LDPE/LLDPE 0 wt%
LDPE/LLDPE Ecologic 1 wt%
LDPE/LLDPE Ecologic 1 wt%
LDPE/LLDPE Ecologic 1 wt%
LDPE/LLDPE Ecologic 5 wt%
LDPE/LLDPE Ecologic 5 wt%
LDPE/LLDPE Ecologic 5 wt%
LDPE/LLDPE Symphony 1 wt%
LDPE/LLDPE Symphony 1 wt%
LDPE/LLDPE Symphony 1 wt%
LDPE/LLDPE Symphony 5 wt%
LDPE/LLDPE Symphony 5 wt%
LDPE/LLDPE Symphony 5 wt%
LDPE/LLDPE Wells 1 wt%
LDPE/LLDPE Wells 1 wt%
LDPE/LLDPE Wells 1 wt%
LDPE/LLDPE Wells 5 wt%
LDPE/LLDPE Wells 5 wt%
LDPE/LLDPE Wells 5 wt%

2.2470
2.2491
2.2438
2.2487
2.2479
2.2464
2.2420
2.2463
2.2449
2.2442
2.2432
2.2486
2.2464
2.2460
2.2447
2.2473
2.2497
2.2434
2.2478
2.2465
2.2483

47

Table A-5. Weight for PET samples (50 °C).
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87

PET 0 wt%
PET 0 wt%
PET 0 wt%
PET Ecologic 1 wt%
PET Ecologic 1 wt%
PET Ecologic 1 wt%
PET Ecologic 5 wt%
PET Ecologic 5 wt%
PET Ecologic 5 wt%
PET Wells 1 wt%
PET Wells 1 wt%
PET Wells 1 wt%
PET Wells 5 wt%
PET Wells 5 wt%
PET Wells 5 wt%

3.0883
3.0828
3.0852
3.0848
3.0825
3.0854
3.0807
3.0832
3.0873
3.0809
3.0850
3.0845
3.0814
3.0803
3.0849

Table A-6. Weight for cellulose samples (50 °C).
R2
R2
R3
S1
S2
S3

Cellulose 0.55g
Cellulose 0.55g
Cellulose 0.55g
Cellulose 1.10g
Cellulose 1.10g
Cellulose 1.10g

0.5567
0.5528
0.5543
1.1008
1.1054
1.1076

48

APPENDIX B: ACCUMULATED GAS MEASUREMENT

Table B-1. Accumulated gas measurement for LDPE/LLDPE 0 wt% (35 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#1
#2
#3
0
0
0
75
77
76
205
205
206
470
488
486
682
693
601
831
872
796
873
892
806
891
907
818
923
939
847
952
974
871
980
1017
909
1012
1049
935
1067
1106
987
1122
1165
1041
1137
1182
1055
1149
1197
1067
1160
1208
1076
1188
1243
1123
1210
1267
1145
1241
1296
1167
1266
1330
1190
1271
1331
1195

49

Average accumulative
gas (mL)

Standard
deviation

0
76
205
481
659
833
857
872
903
932
969
999
1053
1109
1125
1138
1148
1185
1207
1235
1262
1266

0.0
1.0
0.6
9.9
50.2
38.0
45.2
47.4
49.2
54.2
54.9
58.2
60.7
63.0
64.4
65.7
66.8
60.1
61.0
64.7
70.1
68.2

Table B-2. Accumulated gas measurement for LDPE/LLDPE Ecologic 1 wt% (35 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#4
#5
#6
0
0
0
81
81
72
214
205
203
504
502
489
684
687
689
882
893
886
904
910
912
921
929
934
950
954
964
979
990
989
1012
1029
1030
1056
1059
1072
1120
1113
1127
1189
1171
1189
1208
1189
1213
1225
1204
1233
1239
1213
1250
1268
1250
1310
1277
1274
1343
1307
1323
1373
1339
1346
1397
1343
1362
1413

50

Average accumulative
gas (mL)

Standard
deviation

0
78
207
498
687
887
909
928
956
986
1024
1062
1120
1183
1203
1221
1234
1276
1298
1334
1361
1373

0.0
5.2
5.9
8.1
2.5
5.6
4.2
6.6
7.2
6.1
10.1
8.5
7.0
10.4
12.7
15.0
19.0
30.8
39.0
34.4
31.7
36.2

Table B-3. Accumulated gas measurement for LDPE/LLDPE Ecologic 5 wt% (35 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#7
#8
#9
0
0
0
82
86
75
227
241
217
570
544
507
754
679
722
870
801
868
911
821
887
932
848
909
979
888
949
1007
908
987
1037
943
1026
1064
981
1058
1122
1048
1110
1205
1123
1170
1225
1153
1189
1238
1176
1206
1247
1196
1217
1280
1233
1260
1302
1245
1284
1336
1282
1313
1363
1312
1343
1374
1318
1355

51

Average accumulative
gas (mL)

Standard
deviation

0
81
228
540
718
846
873
896
939
967
1002
1034
1093
1166
1189
1207
1220
1258
1277
1310
1339
1349

0.0
5.6
12.1
31.7
37.6
39.3
46.6
43.4
46.4
52.3
51.4
46.3
39.7
41.1
36.0
31.0
25.6
23.6
29.1
27.1
25.7
28.5

Table B-4. Accumulated gas measurement for LDPE/LLDPE Symphony 1 wt% (35 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#10
#11
#12
0
0
0
89
71
87
193
163
207
471
403
452
711
651
692
961
856
847
1025
899
907
1064
929
926
1122
978
960
1160
1004
992
1193
1038
1022
1227
1065
1060
1280
1125
1096
1363
1172
1160
1405
1187
1178
1437
1199
1194
1467
1203
1207
1522
1235
1244
1556
1250
1269
1605
1286
1301
1650
1312
1334
1658
1324
1348

52

Average accumulative
gas (mL)

Standard
deviation

0
82
188
442
685
888
944
973
1020
1052
1084
1117
1167
1232
1257
1277
1292
1334
1358
1397
1432
1443

0.0
9.9
22.5
35.1
30.7
63.4
70.6
78.8
88.8
93.7
94.4
95.0
98.9
113.9
128.5
138.9
151.3
163.2
171.4
180.0
189.1
186.3

Table B-5. Accumulated gas measurement for LDPE/LLDPE Symphony 5 wt% (35 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#13
#14
#15
0
0
0
83
76
81
223
191
203
503
451
446
698
676
666
868
858
856
895
882
861
909
911
885
943
940
921
969
966
956
1022
992
996
1057
1017
1041
1118
1069
1108
1190
1133
1170
1208
1145
1187
1221
1151
1201
1231
1156
1212
1273
1207
1241
1296
1230
1257
1322
1260
1296
1354
1298
1401
1370
1306
1402

53

Average accumulative
gas (mL)

Standard
deviation

0
80
206
467
680
861
879
902
935
964
1003
1038
1098
1164
1180
1191
1200
1240
1261
1293
1351
1359

0.0
3.6
16.2
31.6
16.4
6.4
17.2
14.5
11.9
6.8
16.3
20.1
25.9
28.9
32.1
36.1
39.0
33.0
33.2
31.1
51.6
48.9

Table B-6. Accumulated gas measurement for LDPE/LLDPE Wells 1 wt% (35 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#16
#17
#18
0
0
0
81
82
82
219
215
218
526
517
507
687
712
707
831
900
855
868
928
891
884
947
911
927
982
949
956
1006
988
987
1042
1022
1014
1074
1044
1072
1127
1102
1153
1190
1170
1183
1211
1191
1205
1228
1208
1225
1244
1220
1272
1289
1258
1293
1312
1278
1326
1352
1314
1362
1394
1344
1375
1401
1350

54

Average accumulative
gas (mL)

Standard
deviation

0
82
217
517
702
862
896
914
953
983
1017
1044
1100
1171
1195
1214
1230
1273
1294
1331
1367
1375

0.0
0.6
2.1
9.5
13.2
35.0
30.3
31.6
27.7
25.3
27.8
30.0
27.5
18.5
14.4
12.5
12.7
15.5
17.0
19.4
25.3
25.5

Table B-7. Accumulated gas measurement for LDPE/LLDPE Wells 5 wt% (35 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#19
#20
#21
0
0
0
78
79
74
190
220
194
420
463
449
633
693
639
844
885
851
881
900
892
899
924
918
935
951
955
969
980
984
1008
1013
1012
1034
1046
1052
1085
1106
1107
1131
1150
1173
1140
1171
1190
1146
1186
1204
1151
1196
1217
1188
1235
1250
1208
1235
1270
1244
1269
1315
1287
1302
1342
1299
1305
1352

55

Average accumulative
gas (mL)

Standard
deviation

0
77
201
444
655
860
891
914
947
978
1011
1044
1099
1151
1167
1179
1188
1224
1238
1276
1310
1319

0.0
2.6
16.3
21.9
33.0
21.9
9.5
13.1
10.6
7.8
2.6
9.2
12.4
21.0
25.2
29.7
33.7
32.3
31.1
36.0
28.4
29.0

Table B-8. Accumulated gas measurement for PET 0 wt% (35 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#22
#23
#24
0
0
0
80
84
83
227
228
213
497
498
388
707
688
598
886
879
780
912
930
800
928
950
815
960
973
851
989
1005
890
1015
1038
930
1053
1083
961
1108
1138
1012
1162
1200
1060
1177
1225
1079
1190
1243
1097
1200
1253
1114
1238
1285
1156
1252
1296
1177
1282
1321
1226
1310
1353
1270
1317
1363
1274

56

Average accumulative
gas (mL)

Standard
deviation

0
82
223
461
664
848
881
898
928
961
994
1032
1086
1141
1160
1177
1189
1226
1242
1276
1311
1318

0.0
2.1
8.4
63.2
58.2
59.3
70.4
72.4
67.0
62.3
56.9
63.6
65.8
72.4
74.4
73.9
70.1
65.3
60.2
47.8
41.5
44.5

Table B-9. Accumulated gas measurement for PET Ecologic 1 wt% (35 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#25
#26
#27
0
0
0
82
76
84
208
198
211
430
433
422
670
673
652
871
852
822
884
872
872
904
890
893
925
929
940
944
961
965
982
993
998
1009
1020
1039
1056
1078
1107
1117
1139
1171
1136
1148
1184
1150
1153
1194
1161
1156
1203
1189
1186
1234
1213
1198
1251
1245
1234
1278
1272
1288
1303
1277
1304
1306

57

Average accumulative
gas (mL)

Standard
deviation

0
81
206
428
665
848
876
896
931
957
991
1023
1080
1142
1156
1166
1173
1203
1221
1252
1288
1296

0.0
4.2
6.8
5.7
11.4
24.7
6.9
7.4
7.8
11.2
8.2
15.2
25.6
27.2
25.0
24.6
25.8
26.9
27.3
22.9
15.5
16.2

Table B-10. Accumulated gas measurement for PET Ecologic 5 wt% (35 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#28
#29
#30
0
0
0
79
83
78
204
241
223
469
505
478
691
735
690
848
895
831
873
915
875
894
932
909
928
961
949
957
987
982
989
1015
1016
1026
1054
1051
1080
1112
1133
1133
1168
1218
1145
1188
1243
1153
1204
1263
1158
1217
1277
1185
1248
1325
1205
1276
1343
1237
1315
1372
1286
1340
1388
1292
1348
1403

58

Average accumulative
gas (mL)

Standard
deviation

0
80
223
484
705
858
888
912
946
975
1007
1044
1108
1173
1192
1207
1217
1253
1275
1308
1338
1348

0.0
2.6
18.5
18.7
25.7
33.2
23.7
19.1
16.7
16.1
15.3
15.4
26.7
42.7
49.1
55.0
59.5
70.1
69.0
67.8
51.0
55.5

Table B-11. Accumulated gas measurement for PET Wells 1 wt% (35 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#31
#32
#33
0
0
0
82
80
78
217
216
206
469
461
436
711
691
676
890
872
867
916
907
900
942
925
920
980
959
943
1004
989
971
1032
1016
1010
1082
1055
1044
1141
1112
1091
1183
1170
1149
1200
1188
1170
1215
1200
1188
1226
1210
1202
1256
1251
1248
1274
1278
1274
1300
1311
1313
1332
1367
1347
1341
1377
1360

59

Average accumulative
gas (mL)

Standard
deviation

0
80
213
455
693
876
908
929
961
988
1019
1060
1115
1167
1186
1201
1213
1252
1275
1308
1349
1359

0.0
2.0
6.1
17.2
17.6
12.1
8.0
11.5
18.6
16.5
11.4
19.6
25.1
17.2
15.1
13.5
12.2
4.0
2.3
7.0
17.6
18.0

Table B-12. Accumulated gas measurement for PET Wells 5 wt% (35 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#34
#35
#36
0
0
0
77
75
78
198
210
184
458
425
450
688
675
680
840
844
866
872
893
944
895
910
961
945
937
988
971
963
1015
1003
993
1038
1039
1027
1068
1099
1080
1113
1155
1160
1176
1170
1190
1195
1181
1215
1212
1185
1236
1227
1211
1288
1254
1232
1315
1254
1266
1340
1290
1284
1356
1318
1299
1365
1322

60

Average accumulative
gas (mL)

Standard
deviation

0
77
197
444
681
850
903
922
957
983
1011
1045
1097
1164
1185
1203
1216
1251
1267
1299
1319
1329

0.0
1.5
13.0
17.2
6.6
14.0
37.0
34.6
27.4
28.0
23.6
21.1
16.6
11.0
13.2
18.8
27.2
38.6
43.0
37.8
36.0
33.5

Table B-13. Accumulated gas measurement for manure only (1st run) (35 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#43
#44
#45
0
0
0
81
84
80
194
225
211
403
444
444
652
683
680
831
862
858
867
884
894
883
894
913
898
920
941
928
951
965
955
984
1003
991
1013
1037
1045
1063
1072
1091
1128
1132
1101
1152
1155
1107
1172
1175
1109
1185
1187
1146
1220
1223
1167
1237
1241
1193
1266
1289
1211
1324
1356
1222
1336
1369

61

Average accumulative
gas (mL)

Standard
deviation

0
82
210
430
672
850
882
897
920
948
981
1014
1060
1117
1136
1151
1160
1196
1215
1249
1297
1309

0.0
2.1
15.5
23.7
17.1
16.9
13.7
15.2
21.5
18.7
24.2
23.0
13.7
22.6
30.3
38.4
44.5
43.6
41.6
50.1
76.2
77.1

Table B-14. Accumulated gas measurement for manure only (2nd run) (35 °C).
Accumulative gas (mL)
Day
0
6
12
15
23
26
32
40
50
64
78
100
117
169
209
252

Bioreactor Bioreactor Bioreactor
Q1
Q2
Q3
0
0
0
32
34
29
160
163
154
262
248
247
410
401
356
443
441
392
464
536
461
524
644
578
679
717
650
800
843
780
912
948
902
1044
1066
1011
1164
1151
1133
1217
1191
1178
1262
1244
1238
1289
1274
1273

62

Average accumulative
gas (mL)

Standard
deviation

0
32
159
252
389
425
487
582
682
808
921
1040
1149
1195
1248
1279

0.0
2.5
4.6
8.4
28.9
28.9
42.5
60.1
33.6
32.2
24.2
27.7
15.6
19.9
12.5
9.0

Table B-15. Accumulated gas measurement for cellulose 0.55g (35 °C).
Accumulative gas (mL)
Day
0
6
12
15
23
26
32
40
50
64
78
100
117
169
209
252

Bioreactor Bioreactor Bioreactor
R1
R2
R3
0
0
0
35
33
32
205
204
215
375
412
467
546
626
697
616
701
761
805
780
830
971
952
997
1142
1112
1136
1322
1284
1297
1467
1419
1407
1637
1557
1519
1735
1662
1614
1782
1715
1665
1837
1775
1727
1874
1817
1762

63

Average accumulative
gas (mL)

Standard
deviation

0
33
208
418
623
693
805
973
1130
1301
1431
1571
1670
1721
1780
1818

0.0
1.5
6.1
46.3
75.5
72.9
25.0
22.6
15.9
19.3
31.7
60.2
60.9
58.7
55.1
56.0

Table B-16. Accumulated gas measurement for cellulose 1.10g (35 °C).
Accumulative gas (mL)
Day
0
6
12
15
23
26
32
40
50
64
78
100
117
169
209
252

Bioreactor Bioreactor Bioreactor
S1
S2
S3
0
0
0
35
40
38
245
208
228
483
449
473
756
699
718
836
777
804
961
895
978
1052
1091
1187
1153
1266
1320
1315
1402
1452
1465
1513
1592
1565
1753
1712
1716
1861
1937
1759
1922
1960
1814
1972
1996
1837
1985
2013

64

Average accumulative
gas (mL)

Standard
deviation

0
38
227
468
724
806
945
1110
1246
1390
1523
1677
1838
1880
1927
1945

0.0
2.5
18.5
17.5
29.0
29.5
43.8
69.5
85.2
69.3
64.1
98.9
112.3
106.8
98.9
94.6

Table B-17. Accumulated gas measurement for LDPE/LLDPE 0 wt% (50 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#52
#53
#54
0
0
0
46
44
49
110
105
113
192
177
194
452
387
374
567
482
510
595
511
532
617
535
577
666
565
589
716
605
639
754
641
682
774
669
711
812
707
761
852
758
800
868
773
817
881
787
832
893
798
843
917
827
867
932
827
877
955
855
905
973
872
931
988
885
951

65

Average accumulative
gas (mL)

Standard
deviation

0
46
109
188
404
520
546
576
607
653
692
718
760
803
819
833
845
870
879
905
925
941

0.0
2.5
4.0
9.3
41.8
43.3
43.7
41.0
52.8
56.9
57.2
52.8
52.5
47.1
47.5
47.0
47.5
45.1
52.5
50.0
50.7
52.2

Table B-18. Accumulated gas measurement for LDPE/LLDPE Ecologic 1 wt% (50 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#55
#56
#57
0
0
0
51
41
48
100
95
128
210
195
273
490
365
457
565
435
547
591
480
600
609
506
624
660
544
658
701
595
707
727
642
752
739
674
782
776
719
838
815
760
877
826
774
891
835
784
902
841
793
912
871
818
935
871
818
960
910
845
998
931
869
1027
937
887
1031

66

Average accumulative
gas (mL)

Standard
deviation

0
47
108
226
437
516
557
580
621
668
707
732
778
817
830
840
849
875
883
918
942
952

0.0
5.1
17.8
41.4
64.8
70.4
66.8
64.2
66.4
63.0
57.7
54.4
59.5
58.5
58.6
59.2
59.9
58.6
71.8
76.8
79.6
73.1

Table B-19. Accumulated gas measurement for LDPE/LLDPE Ecologic 5 wt% (50 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#58
#59
#60
0
0
0
49
52
53
103
102
115
132
163
171
157
452
289
157
582
379
230
606
443
290
624
505
400
653
577
515
686
637
652
735
688
716
765
719
804
823
762
886
868
814
909
878
826
928
886
836
943
893
844
984
920
876
1010
944
890
1036
988
923
1066
1018
950
1077
1030
964

67

Average accumulative
gas (mL)

Standard
deviation

0
51
107
155
299
373
426
473
543
613
692
733
796
856
871
883
893
927
948
982
1011
1024

0.0
2.1
7.2
20.6
147.8
212.6
188.6
169.3
129.8
88.1
41.6
27.5
31.2
37.5
41.9
46.1
49.5
54.3
60.1
56.7
58.3
56.8

Table B-20. Accumulated gas measurement for LDPE/LLDPE Symphony 1 wt% (50 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#61
#62
#63
0
0
0
56
47
49
120
107
115
165
267
255
200
477
445
200
550
642
247
568
687
339
583
704
562
621
741
612
657
780
671
686
825
671
711
851
740
748
905
813
787
952
826
801
972
837
812
989
845
822
1001
878
851
1034
887
861
1046
916
888
1074
940
910
1100
948
929
1109

68

Average accumulative
gas (mL)

Standard
deviation

0
51
114
229
374
464
501
542
641
683
727
744
798
851
866
879
889
921
931
959
983
995

0.0
4.7
6.6
55.7
151.5
233.2
227.6
185.9
91.2
87.0
84.9
94.5
93.0
88.7
92.4
95.8
97.4
98.8
100.2
100.3
102.1
98.9

Table B-21. Accumulated gas measurement for LDPE/LLDPE Symphony 5 wt% (50 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#64
#65
#66
0
0
0
48
49
54
150
114
118
380
174
161
640
409
181
753
638
206
767
664
269
785
704
358
822
752
496
869
783
578
897
828
662
914
860
712
942
919
767
983
953
822
996
965
842
1007
974
858
1016
982
870
1044
1005
901
1056
1017
901
1086
1044
934
1104
1073
957
1109
1089
972

69

Average accumulative
gas (mL)

Standard
deviation

0
50
127
238
410
532
567
616
690
743
796
829
876
919
934
946
956
983
991
1021
1045
1057

0.0
3.2
19.7
122.9
229.5
288.4
262.9
226.8
171.6
149.5
120.8
104.6
95.1
85.6
81.5
78.3
76.4
73.9
80.6
78.5
77.5
74.0

Table B-22. Accumulated gas measurement for LDPE/LLDPE Wells 1 wt% (50 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#67
#68
#69
0
0
0
45
47
47
117
212
115
229
462
139
433
612
159
572
728
159
592
745
234
617
766
324
658
803
464
709
865
493
738
910
653
761
935
846
800
969
1041
844
1010
1128
858
1031
1145
871
1046
1160
881
1057
1170
909
1092
1205
909
1119
1223
937
1153
1245
969
1177
1270
971
1197
1281

70

Average accumulative
gas (mL)

Standard
deviation

0
46
148
277
401
486
524
569
642
689
767
847
937
994
1011
1026
1036
1069
1084
1112
1139
1150

0.0
1.2
55.4
166.7
228.2
294.0
262.3
224.9
170.1
186.8
130.9
87.0
123.7
142.7
144.5
145.6
145.6
149.4
160.0
158.1
154.1
160.3

Table B-23. Accumulated gas measurement for LDPE/LLDPE Wells 5 wt% (50 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#70
#71
#72
0
0
0
49
49
50
111
148
187
281
358
397
526
528
592
676
626
639
705
654
664
737
669
676
777
704
722
821
750
766
864
780
801
900
806
816
939
849
851
981
887
885
997
901
901
1008
913
918
1018
922
928
1047
949
956
1060
963
968
1089
992
997
1115
1022
1012
1132
1036
1019

71

Average accumulative
gas (mL)

Standard
deviation

0
49
149
345
549
647
674
694
734
779
815
841
880
918
933
946
956
984
997
1026
1050
1062

0.0
0.6
38.0
59.0
37.5
25.9
27.0
37.4
38.0
37.2
43.7
51.6
51.4
54.9
55.4
53.5
53.8
54.7
54.6
54.6
56.8
60.9

Table B-24. Accumulated gas measurement for PET 0 wt% (50 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#73
#74
#75
0
0
0
47
49
50
113
105
105
195
159
245
365
338
499
522
408
588
572
569
625
597
605
640
648
674
659
687
749
701
731
807
739
746
851
771
799
900
808
842
959
872
850
970
886
856
979
897
861
986
906
879
1015
930
879
1024
930
905
1053
951
926
1076
978
937
1087
984

72

Average accumulative
gas (mL)

Standard
deviation

0
49
108
200
401
506
589
614
660
712
759
789
836
891
902
911
918
941
944
970
993
1003

0.0
1.5
4.6
43.2
86.2
91.1
31.5
22.9
13.1
32.5
41.8
54.8
55.9
60.8
61.6
62.6
63.3
68.7
73.6
75.7
76.2
76.7

Table B-25. Accumulated gas measurement for PET Ecologic 1 wt% (50 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#76
#77
#78
0
0
0
54
48
47
160
118
107
355
258
158
545
494
208
595
583
261
620
622
359
630
642
404
648
695
506
687
749
540
732
793
589
770
830
679
813
885
741
867
932
779
882
944
791
894
954
801
903
961
809
930
990
834
940
1018
844
977
1047
868
999
1069
893
1013
1073
899

73

Average accumulative
gas (mL)

Standard
deviation

0
50
128
257
416
480
534
559
616
659
705
760
813
859
872
883
891
918
934
964
987
995

0.0
3.8
28.0
98.5
181.6
189.5
151.3
134.1
98.4
107.3
104.7
76.0
72.0
76.8
77.0
77.1
76.7
78.7
87.2
90.2
88.6
88.4

Table B-26. Accumulated gas measurement for PET Ecologic 5 wt% (50 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#79
#80
#81
0
0
0
50
51
52
122
105
112
217
150
312
489
340
487
599
470
717
619
509
734
638
528
753
673
572
789
708
634
834
774
675
869
804
712
890
835
747
923
882
788
957
897
799
969
910
809
980
917
815
991
944
845
1006
954
854
1016
977
883
1035
1001
903
1054
1013
905
1074

74

Average accumulative
gas (mL)

Standard
deviation

0
51
113
226
439
595
621
640
678
725
773
802
835
876
888
900
908
932
941
965
986
997

0.0
1.0
8.5
81.4
85.5
123.5
112.5
112.5
108.6
101.1
97.0
89.0
88.0
84.7
85.3
86.0
88.4
81.2
81.7
76.7
76.6
85.6

Table B-27. Accumulated gas measurement for PET Wells 1 wt% (50 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#82
#83
#84
0
0
0
46
47
46
107
107
170
143
134
482
149
173
658
149
218
700
291
421
721
380
492
738
483
600
738
613
698
758
790
767
794
947
814
828
1127
874
874
1238
917
911
1268
933
920
1293
946
926
1312
956
931
1373
983
951
1373
983
951
1426
1013
970
1472
1053
989
1487
1066
999

75

Average accumulative
gas (mL)

Standard
deviation

0
46
128
253
327
356
478
537
607
690
784
863
958
1022
1040
1055
1066
1102
1102
1136
1171
1184

0.0
0.6
36.4
198.4
287.2
300.2
220.5
183.1
127.6
72.9
14.6
73.1
146.1
187.1
197.3
206.4
213.1
234.9
234.9
251.8
262.3
264.5

Table B-28. Accumulated gas measurement for PET Wells 5 wt% (50 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#85
#86
#87
0
0
0
38
44
50
98
169
113
170
269
126
425
454
156
634
508
156
670
526
259
707
541
326
747
571
456
818
610
581
847
648
730
871
682
819
905
723
903
941
768
991
955
784
1014
964
795
1032
971
805
1045
991
836
1083
991
836
1107
1013
869
1145
1024
890
1184
1041
904
1200

76

Average accumulative
gas (mL)

Standard
deviation

0
44
127
188
345
433
485
525
591
670
742
791
844
900
918
930
940
970
978
1009
1033
1048

0.0
6.0
37.4
73.2
164.3
247.7
208.5
191.0
146.6
129.3
100.0
97.6
104.5
117.0
119.5
122.0
122.9
124.8
136.0
138.0
147.2
148.1

Table B-29. Accumulated gas measurement for manure only (1st run) wt% (50 °C).
Accumulative gas (mL)
Day
0
6
12
23
35
50
62
66
75
84
93
105
126
160
174
188
203
237
260
315
387
464

Bioreactor Bioreactor Bioreactor
#94
#95
#96
0
0
0
50
39
42
110
83
104
246
187
244
412
482
524
553
542
615
583
590
635
606
629
650
643
668
700
698
709
740
740
735
781
772
761
798
809
792
834
844
818
856
863
822
865
880
825
873
895
828
881
923
851
905
941
859
905
963
881
920
980
903
942
991
912
962

77

Average accumulative
gas (mL)

Standard
deviation

0
44
99
226
473
570
603
628
670
716
752
777
812
839
850
859
868
893
902
921
942
955

0.0
5.7
14.2
33.5
56.6
39.4
28.2
22.0
28.6
21.8
25.2
19.0
21.1
19.4
24.3
29.9
35.3
37.5
41.1
41.0
38.5
40.0

Table B-30. Accumulated gas measurement for manure only (2nd run) wt% (50 °C).
Accumulative gas (mL)
Day
0
6
12
15
23
26
32
40
50
64
78
100
117
169
209
252

Bioreactor Bioreactor Bioreactor
N1
N2
N3
0
0
0
272
275
273
511
512
511
611
590
603
679
658
673
699
676
691
736
696
715
765
716
742
807
750
789
868
815
850
916
865
925
976
920
984
1027
957
1023
1038
984
1044
1089
1044
1064
1111
1075
1079

78

Average accumulative
gas (mL)

Standard
deviation

0
273
511
601
670
689
716
741
782
844
902
960
1002
1022
1066
1088

0.0
1.5
0.6
10.6
10.8
11.7
20.0
24.5
29.1
27.0
32.4
34.9
39.3
33.0
22.5
19.7

Table B-31. Accumulated gas measurement for cellulose 0.55g (50 °C).
Accumulative gas (mL)
Day
0
6
12
15
23
26
32
40
50
64
78
100
117
169
209
252

Bioreactor Bioreactor Bioreactor
O1
O2
O3
0
0
0
382
390
380
692
628
700
903
828
931
1051
909
1061
1086
941
1094
1112
984
1122
1162
1014
1165
1210
1062
1210
1279
1137
1280
1359
1208
1349
1443
1274
1421
1495
1325
1470
1592
1435
1600
1646
1480
1660
1669
1497
1687

79

Average accumulative
gas (mL)

Standard
deviation

0
384
673
887
1007
1040
1073
1114
1161
1232
1305
1379
1430
1542
1595
1618

0.0
5.3
39.5
53.3
85.0
86.1
77.0
86.3
85.4
82.3
84.4
91.9
91.8
93.0
100.1
104.9

Table B-32. Accumulated gas measurement for cellulose 1.10g (50 °C).
Accumulative gas (mL)
Day
0
6
12
15
23
26
32
40
50
64
78
100
117
169
209
252

Bioreactor Bioreactor Bioreactor
P1
P2
P3
0
0
0
374
370
391
594
765
708
872
1094
991
1043
1279
1178
1103
1341
1245
1220
1471
1358
1289
1539
1460
1370
1608
1526
1480
1695
1626
1570
1794
1710
1641
1872
1780
1710
1928
1844
1783
1968
1877
1863
2020
1932
1915
2045
1959

80

Average accumulative
gas (mL)

Standard
deviation

0
378
689
986
1167
1230
1350
1429
1501
1600
1691
1764
1827
1876
1938
1973

0.0
11.2
87.1
111.1
118.4
119.7
125.7
127.8
120.9
109.8
113.2
116.3
110.0
92.5
78.7
66.1

APPENDIX C: SPIKING GAS MEASUREMENT DATA

Table C-1. Spiking gas measurement for bioreactors at 35 °C.
Day
0
5
11
20
50

#1
0
5
58
129
140

#9
0
15
69
131
173

#14
0
0
61
126
136

Bioreactors
#21
0
9
73
142
159

r# 28
0
3
49
125
160

#36
0
0
70
145
171

#44
0
0
40
130
165

Table C-2. Spiking gas measurement for bioreactors at 50 °C.
Day
0
5
11
20
50

#53
0
89
138
172
183

#60
0
66
141
184
199

#64
0
69
156
178
197

Bioreactors
#71
#73
0
0
68
59
133
111
173
170
185
193

81

#81
0
66
146
165
182

#85
0
76
157
180
201

#94
0
52
99
164
187

APPENDIX D: ANOVA TABLES

Table D-1. ANOVA table for LDPE/LLDPE samples and controls at 35 °C.
Source
SS
Columns 1541352
Error
122760
Total
1664112

df
10
22
32

MS
F
154135.2 27.62279
5580

Prob>F
3.63E-10

Table D-2. ANOVA table for PET samples and controls at 35 °C.
Source
SS
Columns 1524829
Error
45592.67
Total
1570422

df
8
18
26

MS
F
190603.7 75.25039
2532.926

Prob>F
2.98E-12

Table D-3. ANOVA table for LDPE/LLDPE samples and controls at 50 °C.
Source
SS
Columns 3223681
Error
146648
Total
3370329

df
10
22
32

MS
F
322368.1 48.36137
6665.818

Prob>F
1.23E-12

Table D-4. ANOVA table for PET samples and controls at 50°C.
Source
SS
Columns 2968047
Error
260611.3
Total
3228658

df
8
18
26

MS
F
371005.8 25.62477
14478.41

82

Prob>F
2.55E-08

APPENDIX E: BOXPLOTS

Figure E-1. Boxplots for LDPE/LLDPE samples and controls at 35 °C.

In the box plot, the central line is the median. The edges of the box are the 25th and 75th
percentiles. The whiskers are extended to include the most extreme data points. Two medians are

83

significantly different (α = 0.05) if their intervals (from the lower the upper extremes of the
notches) do not overlap.

Figure E-2. Boxplots for PET samples and controls at 35 °C.

84

Figure E-3. Boxplots for LDPE/LLDPE samples and controls at 50 °C.

85

Figure E-4. Boxplots for PET samples and controls at 50 °C.

86

APPENDIX F: MATLAB CODE FOR PLOTTING MAIN EXPERIMENT DATA

clear
close all
clc
addpath(fullfile(pwd,'export_fig'))
format compact
cmap = hsv(12);
%% Interface for choosing dataset:
fprintf('Choose a dataset:\n')
fprintf(' 1. LDPE 35C\n')
fprintf(' 2. LDPE 50C\n')
fprintf(' 3. PET 35C\n')
fprintf(' 4. PET 50C\n')
choice = input('Enter a number: ');
switch choice
case 1
fileName = 'LDPE 35C';
case 2
fileName = 'LDPE 50C';
case 3
fileName = 'PET 35C';
case 4
fileName = 'PET 50C';
otherwise
halt
end
%% Plotting main graph:
load(fullfile(pwd,'Data',[fileName '.mat']))
nOfSamples = size(data,1);
figure('name',fileName);
hold on
box on
for sampleNumber = 1:nOfSamples
data{sampleNumber,3} = zeros(0,2);
for n = 1:(numel(data{sampleNumber,2}(:,1))/3) % Iterate through each three replicates
a = 3*(n-1) + 1; % Position of the first replicate
b = 3*(n-1) + 3; % Position of the last replicate
data{sampleNumber,3}(end+1,1) = data{sampleNumber,2}(a,1); % Copy the dates
87

data{sampleNumber,3}(end,2) = mean(data{sampleNumber,2}(a:b,2));
data{sampleNumber,3}(end,3) = std(data{sampleNumber,2}(a:b,2));
end
H1(sampleNumber) = errorbar(data{sampleNumber,3}(:,1),data{sampleNumber,3}(:,2),...
data{sampleNumber,3}(:,3),'LineStyle','-','Color',...
cmap(sampleNumber,:),'LineWidth',0.9);
end
hold off
set(gcf, 'Position', [100 10 700 850])
xlim([0,500])
ylim([0,2400])
set(gca,'YTick',0:400:2400)
xlabel('Time, d')
ylabel('Accumulated gas, mL')
H1_legend = legend(H1,data(:,1),'location','SouthEast');
set(H1_legend, 'Box', 'off')
set(H1_legend,'FontSize',10);
set(gcf, 'Color', 'w');
%% Save figure as a high quality png:
export_fig(fullfile(pwd,'Images',sprintf('%s.png',fileName)),'-png');

88

APPENDIX G: MATLAB CODE FOR PLOTTING SPIKING EXPERIMENT DATA

clear
close all
clc
addpath(fullfile(pwd,'export_fig'))
format compact
cmap = hsv(12);
%% Interface for choosing dataset:
fprintf('Choose a dataset:\n')
fprintf(' 1. LDPE 35C\n')
fprintf(' 2. LDPE 50C\n')
fprintf(' 3. PET 35C\n')
fprintf(' 4. PET 50C\n')
choice = input('Enter a number: ');
switch choice
case 1
fileName = 'LDPE 35C';
case 2
fileName = 'LDPE 50C';
case 3
fileName = 'PET 35C';
case 4
fileName = 'PET 50C';
otherwise
halt
end
%% Plotting spiking data:
load(fullfile(pwd,'Data',[fileName ' spike.mat']))
nOfSamples = size(spikedata,1);
legend_string = cell(0,1);
hold on
box on
for sampleNumber = 1:nOfSamples
if ~isempty(spikedata{sampleNumber,1})
plot(spikedata{sampleNumber,2}(21:end,1),...
spikedata{sampleNumber,2}(21:end,2),'-',...
'Color',cmap(sampleNumber,:),'LineWidth',0.9);
legend_string{end+1,1} = spikedata{sampleNumber,1};
end
89

end
hold off
set(gcf, 'Position', [100 10 700 500])
xlim([387,514])
xlabel('Time, d')
ylabel('Accumulated gas, mL')
H1_legend = legend(legend_string,'location','SouthEast');
set(H1_legend, 'Box', 'off')
set(H1_legend,'FontSize',10);
set(gcf, 'Color', 'w');
%% Save figure as a high quality png:
export_fig(fullfile(pwd,'Images',sprintf('%s spike.png',fileName)),'-png');

90

APPENDIX H: MATLAB CODE FOR STATISTICAL ANALYSIS

clear
close all
clc
addpath(fullfile(pwd,'export_fig'))
format longG
format compact
%% Interface for choosing dataset:
fprintf('Choose a dataset:\n')
fprintf(' 1. LDPE 35C\n')
fprintf(' 2. LDPE 50C\n')
fprintf(' 3. PET 35C\n')
fprintf(' 4. PET 50C\n')
choice = input('Enter a number: ');
switch choice
case 1
fileName = 'LDPE 35C';
case 2
fileName = 'LDPE 50C';
case 3
fileName = 'PET 35C';
case 4
fileName = 'PET 50C';
otherwise
halt
end
%% Loading data:
load(fullfile(pwd,'Data',[fileName '.mat']))
nOfSamples = size(data,1);
group = cell(0,1);
% Import data from day 464 from main experiment:
X = zeros(0,nOfSamples);
for sampleNumber = 1:nOfSamples-3
X(1,sampleNumber) = data{sampleNumber,2}(64,2);
X(2,sampleNumber) = data{sampleNumber,2}(65,2);
X(3,sampleNumber) = data{sampleNumber,2}(66,2);
group{sampleNumber,1} = data{sampleNumber,1};
end

91

% Import data from day 252 from positive control experiment:
for sampleNumber = nOfSamples-2:nOfSamples
X(1,sampleNumber) = data{sampleNumber,2}(46,2);
X(2,sampleNumber) = data{sampleNumber,2}(47,2);
X(3,sampleNumber) = data{sampleNumber,2}(48,2);
group{sampleNumber,1} = data{sampleNumber,1};
end
%% ANOVA:
% p: p-value.
% table: ANOVA table.
% stats: structure stats used to perform a follow-up multiple comparison test.
% Note: Notches in the boxplot provide a test of group medians.
[p,table,stats] = anova1(X,group);
fprintf('\nANOVA table:\n')
disp(table)
set(gcf, 'Color', 'w');
set(gcf, 'Position', [50 10 700 700])
export_fig(fullfile(pwd,'Images',sprintf('boxplot %s.png',fileName)),'-png');
%% Multiple comparison test using Tukey's HSD:
% 'alpha' = 0.05: Set alpha value to be 0.05.
% 'ctype' = 'hsd': Use Tukey's honestly significant difference criterion.
% 'estimate' = 'anova2': Either 'column' (the default) or 'row' to compare
% column or row means.
figure
[c,m,h,group] = multcompare(stats,'alpha',0.05,'ctype','hsd','estimate','anova2');
set(gcf, 'Color', 'w');
set(gcf, 'Position', [780 10 700 700])
export_fig(fullfile(pwd,'Images',sprintf('HSD %s.png',fileName)),'-png');
% Mean estimates and the standard errors:
fprintf('\nMean estimates and the standard errors:\n')
fprintf(' [Sample]
[Estimates]
[SE]\n');
disp([group num2cell(m)])
% Display the comparison results:
for row = 1:size(c,1);
if 0>c(row,3) & 0<c(row,5)
c(row,6) = 0;
else
c(row,6) = 1;
end
end
fprintf('\nDisplay the comparison results:\n')
92

fprintf(' [Sample 1] [Sample 2] [lower limit of CI]
[Sig. dif.?]\n');
disp([group(c(:,1)),group(c(:,2)),num2cell(c(:,3:6))])

93

[est. dif. in means] [upper limit of CI]

APPENDIX I: MAXIMUM THEORETICAL GAS EVOLUTION FORMULA

Manure was mixed with water to achieve a total solid content of 50 g/L. Chemical
Oxygen Demand (COD) of manure was measured to be 74.1 g/L. Since the same manure was
used to feed the bioreactor where the inoculum came from, it was assumed that the COD of the
inoculum was approximately 74.1 g/L. The COD reduction rate of manure/inoculum was
assumed to be 30%. The COD reduction rate of cellulose was assumed to be 100%.
Stoichiometrically, 1 g of COD reduction is converted into 0.395 L methane at 35°C, 1 atm [66].
Biogas produced was assumed to be 60% CH4 and 40% CO2.

0.395 ‫ܪܥ ܮ‬ସ
1݃ ‫ ܦܱܥ‬ൈ ሺܸ௠௔௡௨௥௘ ൅ ܸ௜௡௢௖௨௟௨௠ ሻ
%ܸ௠௘௧௛௔௡௘ /ܸ௧௢௧௔௟

݇ ൈ ‫ ܦܱܥ‬ൈ %‫ ݊݋݅ݐܿݑ݀݁ݎ_ܦܱܥ‬ൈ
ܸ௚௔௦ ൌ

Vgas: total gas produced.
k: conversion factor based on ideal gas law (k = 1 if at 35 °C; k = 1.04868 if at 50 °C).
COD: COD of manure and inoculum (74.1 g/L).
%COD_reduction: percentage of COD reduction (30%).
Vmanure: Volume of manure (0.075 L).
Vinoculum: Volume of inoculum (0.0075 L).
%Vmethane/Vtotal: percentage of methane in total gas volume (60%).

94

APPENDIX J: WEIGHT OF MANURE

Table J-1. Manure mixture for main experiment.

Replicates
1
2
3
Average

Weight of
aluminum pan,
g
1.2752
1.279
1.2966

Weight of wet
manure and pan,
g
14.6424
20.4102
20.3788

Weight of dry
manure and pan,
g
3.4459
4.3647
4.2399

Weight of wet
manure needed, g
3079.0
3100.0
3241.6
3140.2

Note: Ten liters of 5% (w/v) manure was prepared by mixing 3140.2 g wet manure and adding
DI water to the required volume.

Table J-2. Manure mixture for positive control experiment.

Replicates
1
2
3
Average

Weight of
aluminum pan,
g
1.3272
1.3317
1.3421

Weight of wet
manure and pan,
g
12.9255
12.1992
13.8742

Weight of dry
manure and pan,
g
3.0934
2.9245
3.2469

Weight of wet
manure needed, g
328.34
341.14
328.96
332.82

Note: A liter of 5% (w/v) manure was prepared by mixing 332.82g wet manure and adding DI
water to the required volume.

95

APPENDIX K: AUTOCAD DRAWINGS OF GAS MEASURING APPARATUS

Figure K-1. Dimensions of the components of the gas measuring apparatus.

96

APPENDIX L: GAS MEASUREMENT FOR ORIGINAL POSITIVE CONTROLS

Table L-1. Accumulated gas measurement for starch (original positive control) (35 °C).
Accumulative gas (mL)
Day
6
12
23
35
50

Bioreactor Bioreactor Bioreactor
#37
#38
#39
101
103
106
149
158
155
229
227
235
229
252
260
234
277
260

Average accumulative
gas (mL)

Standard
deviation

103
154
230
247
257

2.5
4.6
4.2
16.1
21.7

Table L-2. Accumulated gas measurement for cellulose (original positive control) (35 °C).
Accumulative gas (mL)
Day
6
12
23
35
50

Bioreactor Bioreactor Bioreactor
#40
#41
#42
85
82
87
132
167
164
187
228
235
217
228
265
252
253
295

Average accumulative
gas (mL)

Standard
deviation

85
154
217
237
267

2.5
19.4
25.9
25.1
24.5

Table L-3. Accumulated gas measurement for starch (original positive control) (50 °C).
Accumulative gas (mL)
Day
6
12
23
35
50

Bioreactor Bioreactor Bioreactor
#88
#89
#90
90
76
95
140
154
183
140
165
218
180
195
238
220
195
238

97

Average accumulative
gas (mL)

Standard
deviation

87
159
174
204
218

9.8
21.9
39.8
30.1
21.6

Table L-4. Accumulated gas measurement for cellulose (original positive control) (50 °C).
Accumulative gas (mL)
Day
6
12
23
35
50

Bioreactor Bioreactor Bioreactor
#91
#92
#93
65
63
69
122
157
102
200
245
124
280
310
173
308
340
208

98

Average accumulative
gas (mL)

Standard
deviation

66
127
190
254
285

3.1
27.8
61.2
72.0
68.9

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