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3 1293 010163

This is to certify that the

thesis entitled

Studies on the Aeiooic
Biodegradation of Polymers

presented by

Juli Hanna David

has been accepted towards fulfillment
of the requirements for

M. 5. degree in CHE

Major professor 2

 

Date 8/4/94

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

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Michigan State
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MSU IoAnNIirmntlvo Action/Equal Oppomnly intuition

 

STUDIES ON THE AEROBIC BIODEGRADATION
OF POLYMERS
By

Juli Hanna David

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

DEPARTMENT OF CHEMICAL ENGINEERING

l 994

ABSTRACT

STUDIES ON THE AEROBIC BIODEGRADATION
OF POLYMERS
By

Juli Hanna David

From an environmental perspective, biodegradable polymers offer an attractive
alternative to traditional petroleum based nonbiodegradable polymers. In this
work, a study was conducted on the aerobic biodegradation of polymers in the
presence of municipal sewage sludge. The extent of biodegradation was quantified
systematically based on percentage carbon conversion to C02. Commercial
polymers like PCL, PHB/V and 9-11 acid were found to be potentially
biodegradable, whereas PVOH, EVOH and Acrylic coating showed little
biodegradation. Focus was directed towards cellulose and starch esters which are
potential cost effective biodegradable plastics. Starch propionates of different
degrees of substitution (DS) showed a decrease in biodegradability with increasing
DS. Two cellulose acetate (CA) products when studied with respect to
morphological effects showed appreciable difference in biodegradation between an
open honeycomb morphology and a closed, smooth morphology. These results
suggest that enzyme accessibility of the glycosidic linkage may be an important
factor governing CA biodegradation , and calls for further investigation.

This thesis is dedicated to

my parents, Jimmy, Jessy and James.

ACKNOWLEDGMENTS

It would take an entire chapter to appropriately acknowledge all the help I
have received during this work. Suffice it to say that I owe it all to God above and
to my colleagues and friends for seeing me through all the ups and downs involved
in my research work. However, certain people should not go unacknowledged. I
would like to thank my advisor, Dr. Ramani Narayan for his guidance and help in
molding my thinking processes for research work, Dr. Long Le and other staff in
the Department of Chemistry for the NMR work, Dr. S. Flegler at Electron Optics
Center for the SEM work, Dr. S. Bloembergen and my colleagues from the
Biomaterials Group at MBI for help with the biodegradation experiments, Dr. M.
R. Natarajan and Dr. C. A. Reddy for help in the microbiology involved, James
Femandes for my oral defence presentation and all my friends at the Composites
Centre for helping me in their own ways.

On a personal note, the support of my family from across thousands of

miles, and James through all this work has been invaluable and inexpressible.

TABLE OF CONTENTS

Chapter
LIST OF TABLES
LIST OF FIGURES

1 INTRODUCTION
1.1 Objective

I .1 .I Biodegradation - Measurement Perspective
1.1.2 Biodegradation - Materials Perspective

1.2 Summary of chapters
2 BIODEGRADABLE MATERIALS
2.1 Waste management Infrastructure
2.2 Plastic Waste Management
2.3 Rationale for biodegradable polymers
2.4 Biodegradable plastics - Regulatory actions
2.5 Biodegradable materials in the industry
2.6 Environmentally Degradable Plastics - Definitions

2.6.1 General Definitions
2.6.2 Specific Definitions ._

3 MEASUREMENT OF BIODEGRADATION

3.1 General Methods of Measurement

V

Page

10
10
12

12
13

l4

l4

v1
3.1.1 Fungal Tests
3.1.2 Soil Burial Tests
3.1.3 Composting experiments
3.1.4 Enzymatic Assays
3.1.5 Liquid Culture experiments

3.2 Selected measurement process description

3 .2.1 Scope

3.2.2 Inoculum Test Organisms
3.2.3 CHN Analysis of test material
3.2.4 Reagents and Test solution
3.2.5 Experiemntal Setup

3.2.6 Measurement of absorbed C 02
3.2.7 The autotitrator

3.2.8 Calculations

4 THE BIODEGRADATION PROCESS

4.1 The basic biodegradation equation

4.2 Ananlysis of the biodegradation process

4.2.] The carbon Balance
4.2.2 Carbon mineralised to C 02

4.3 Factors affecting the biodegradation process

4.3.] Experimental factors
43.2 Material Physical factors
4.3.3 Microbiological factors
43 .4 Chemical composition

4.4 Limiting parameters in the biodegradation equation
4.5 Application to experimental rates of polymer systems

4.6 Effect of Agitation speeds

4.6.1 Experimentals
4.6.2 Results and Discussion

14
15
15
l6
16

19
19
19
20
20
21
25
29
3O
32
32
32

32
33

33
33
35
36
37
38
39
39

39
44

vii
4.7 Effect of acclimatisation of microbial inoculum

4.7.] Materials tested

4.7.2 Preparation of inoculum
4.7.3 Results and discussion

5 BIODEGRADATION OF COMMERCIAL POLYMERS
5.1 Introduction
5.2 Polymers Tested

5.2.1 PHB/V

5.2.2 Poly Caprolactone

5.2.3 Unmodified Corn Starch (Hylon 7)
5.2.4 PCL/Starch Blend

5.2.5 Polyvinyl alcohol (PVOH)

5.2.6 Ethylene vinyl alcohol (E VOH)

5 .2 .7 Glycerol Triacetate

5.2.8. Acrylic Coating

5.2.8 9-1 I Acid

5.3 Experimentals

5.3.1 Inoculums and Test Solutions
5.3.2 Results and Discussion

6 CELLULOSE AND ST ARCH ESTERS

6.1 Background

6.2 Theoretical
6.2.] Structure of Starch
6.2.2 Structure of Cellulose
6.2.3 Structure of Cellulose and Starch Esters

6.3 Experimental Characterization of Cellulose and Starch Esters
6.3.1 Degree of Substitution

6.3.1.1 Determination of DS by the method of titration
6.3.1.2 Determination of DS by proton NMR

44

44
45
46

51

51

51

51
53
53
54
54
55
55
55
55

56

56
66

69
69
70
7O
72
73
74
74

75
76

viii
6.3.2 Position of Substitution
6.4 Correlation of Biodegradability with Position and Degree of
Substitution

7 STARCH ESTERS

7.1 Background

7.2 Synthesis of the Modified Starches

7.3 Biodegradation of Starch Propionates with varying DS
7.3.] Materials Tested
7.3.2 Inoculum and Test Solution
7.3.3 Results and Discussion
7.3.4 Conclusions
7.3 5 Limitations of the above Data

8 CELLULOSE AND CELLULOSE ACETATES

8.1 Background

8.2 Structure of Cellulose Acetate

8.3 Cx - C1 Mechanism of Cellulose Biodegradation

8.4 CA Biodegradation Literature

8.5 CA Biodegradation - Experimental
85.1 Materials Tested
85 .2 Inoculum and Test Solution
8.5 .3 Results

8.6 Effect of Morphology - Literature

8.7 CA with Defined Morphology - Experimentals
8.7.] Preparation of CA samples with Defined Morphology

8.7.2 Inoculum and Test Solution
8.7.3 Biodegradation Results

81
88
89
89
90
93
93
93
96
100
101
102
102
103
103
106
108
108
111
111
117
119
119

119
125

ix
8.8 Discussion

8.9 Proposed Mechanism for CA Biodegradation

8.10 Proposed Kinetic Model for the Aerobic Biodegradation
of Cellulose

8.10.1 Assumptions

8.10.2 Competitive and Non-competitive Inhibition
8.10.3 Mechanism of Degradation

8.10.4 Description of Model

8.105 Development of the Model

9 CONCLUSIONS AND RECOMMENDATIONS

BIBLIOGRAPHY

125
126
128
127
129
130
132
134
139

143

Table 2.1

Table 4.1

Table 4.2

Table 5.1

Table 7.1

Table 8.1

Table 8.2

Table 8.3

LIST OF TABLES

Current Status of the US Biodegradables industry
Summary of biodegradation results

Standard deviations between replicates

Summary of biodegradation results for all setups
Summary of biodegradation results

Material Properties of the CA samples
Differences between the two cellulose acetates

Biodegradation results - Percentage C converted to CO;
with time

11

40

46

66

100

109

117

125

Figure 2.1
Figure 2.2
Figure 3.1
Figure 3.2
Figure 3.3
Figure 3.4
Figure 3.5
Figure 3.6
Figure 4.1
Figure 4.2

Figure 4.3

Figure 4.4
Figure 4.5
Figure 4.6
Figure 5.1

Figure 5.2

LIST OF FIGURES

Materials in MSW by volume (Cubic yards)

Nature’s Carbon Cycle

Summary of the test method

Experimental Setup for Liquid Culture Experiments
Computation of C02 evolution from test flasks
Titration Curve for the carbonate system

The Orion 960 Autochemistry System

Change in mV vs ml of titrant added for the autotitrator
Percentage conversion of PCL carbon to C02

Rate of PCL carbon converted to C02 with time

Relation between speed of agitation and percentage C
conversion to C02

Effect of inoculum acclimatisation on CA-398—3
Effect of inoculum acclimatisation on Modified starch
Effect of inoculum acclimatisation on CA-398-30
Structure of poly(beta-hydroxy alkanoate)

Structure of PCL

xi

19

22

24

26

28

30

41

42

43

47

48

49

52

53

Figure 5.3
Figure 5.4

Figure 5.5

Figure 5.6

Figure 5.7

Figure 5.8

Figure 5.9

Figure 5.10

Figure 5.11

Figure 5.12

Figure 6.1
Figure 6.2
Figure 6.3
Figure 6.4
Figure 6.5
Figure 6.6

Figure 6.7

xii

Chemical structure of Poly vinyl alcohol
Structure of Glycerol niacetate

Percentage C converted to CO; with time for AquaCoat584F
and 9-1 lacid

Percentage initial C converted to CO; with time for Glycerol
tri-acetate

Percentage of initial polymeric C converted to CO; with time
for various commercial polymers

Rate of initial polymeric C conversion to CO; with time for
various commercial polymers

Initial polymeric carbon converted to CO; with time for the
50/50 PCL/starch blend

Percentage of initial polymeric C converted to CO; with time
for various commercial polymers (no blank correction)

Rate of initial polymeric C converted to CO; with time for
various commercial polymers (no blank correction)

Initial polymeric carbon converted to CO; with time for the
50/50 PCL/starch blend (no blank correction)

Structure of unmodified starch (Amylose fraction)

Structure of unmodified starch (Amylopectin)

Structure of cellulose

Position of OH groups on the monomer unit of cellulose/starch
Structure of a triacetate monomer unit of cellulose or starch
Proton NMR of Cellulose acetate without TFA

Proton NMR of Starch propionate without TFA

54

55

58

59

60

61

62

63

64

65

71

71

72

73

74

78

78

Figure 6.8
Figure 6.9
Figure 6.10
Figure 6.11
Figure 6.12
Figure 6.13
Figure 6.14
Figure 6.15
Figure 7.1
Figure 7.2
Figure 7.3
Figure 7.4
Figure 7.5

Figure 7.6

Figure 7.7

Figure 7.8

Figure 8.1

Figure 8.2

Figure 8.3

xiii

Proton NMR of CA with TFA

Proton NMR of Starch propionate with TFA

C13 NMR of unmodified Starch

C13 NMR of starch propionate of DS 3.0

C13 NMR of starch acetate of D820

C13 NMR of starch propionate D81 .0 - Carbonyl peaks
C13 NMR of starch propionate DSl.7 - Carbonyl peaks
C13 NMR of cellulose acetate D825

Glass Reactor Assembly for synthesis of modified starches
Proton NMR of Starch propionate DS = 1.0

Proton NMR of Starch propionate DS = 1.5

Proton NMR of Starch propionate DS = 1.7

Proton NMR of Starch propionate DS = 2.76

Percentage of initial polymeric C converted to CO; with
time for starch propionates of varying DS

Percentage of initial C converted to C02 with time for starch
propionates of varying DS (Cellulose excluded)

Relation between biodegradation and degree of substitution

Structure of Cellulose acetate - random copolymer of
monoacetate, diacetate, triacetate and unsubstituted units

C, - C1 mechanism of enzymatic hydrolysis

Summary of the conflicting reports on CA biodegradation

79

79

82

83

84

86

86

87

91

94

94

95

95

97

98

99

104

105

107

Figure 8.4
Figure 8.5
Figure 8.6
Figure 8.7

Figure 8.8

Figure 8.9

Figure 8.10
Figure 8.11
Figure 8.12
Figure 8.13

Figure 8.14

Figure 8.15

Figure 8.16
Figure 8.17
Figure 8.18

Figure 8.19

Figure 8.20

Figure 8.21

xiv

Proton NMR of CA-398-30

Proton NMR of JLF-68

Percentage of initial polymeric C converted to C02 with time

Rate of initial polymeric C conversion to C02 with time

Percentage of CA carbon converted to CO; with time (no blank

correction)

SEM micrographs of CA-398-30 at 3000K

SEM micrographs of JLF-68 at 3000X
Preparation of samples with defined morphology
Proton NMR of precipitated CA-398-30

Proton NMR of precipitated JLF-68

SEM micrograph of precipitated CA-398-30 at SOOX
and 3000X

SEM micrograph of precipitated JLF-68 at 500x and 3000X

CA-carbon conversion to C02 with time for the precipitated
CAs

CA carbon conversion to C02 for the precipitated CAs (no
blank correction)

Proposed mechanism for aerobic enzymatic degradation of
CA

Competitive and Non-competitive inhibition
Process Schematic for the batch reactor

Reaction mechanisms for E and BB

110

110

112

113

114

115

116

118

120

120

121

122

123

124

126

129

133

134

CA

C/N
DS
EVOH

PCL
PHB/V
PVOH
SP
SEM

LIST OF ABBREVIATIONS

Cellulose acetate of DS=2.5

Polymeric carbon

Carbon / Nitrogen ratio

Degree of Substitution
Ethylene vinyl alcohol
Municipal Sewage Sludge
Nuclear Magnetic Resonance
PolyCaprolactone

Poly hydroxy butyrate/valerate
Polyvinyl alcohol

Starch Propionate

Scanning Electron Microscopy

XV

Chapter I

 

INTRODUCTION

 

Today’s packaging materials are typically designed for utility and application but
with little emphasis on disposability and recyclability. A growing trend is towards
material redesign to produce products with increased environmental compatibility.
A few examples of national and international inclination towards this trend are
listed below [Narayan (1993)].
0 Designing parts that can be disassembled and then recycled, reused, or
incinerated (SPE ANTEC’92 - Environmental Management Forum).
0 German automakers striving to produce recyclable cars - the goal is to
have less than 5% of an automobiles volume going into a landfill (R&D
Magazine, May ‘92)
0 Legislative trends towards designing for disposability and resource
conservation.
0 Environmental Management Standards (ISO - SAGE activities; similar
to ISO 9000 quality assurance standards)
0 International Chamber of Commerce (ICC) charter for the business
development“ Sustainable Environment”
These trends have brought into focus the concept of “degradable” materials - i.e.,
materials that are able to disintegrate in the waste stream in an environmentally
sound manner. In order to integrate material design with material disposability for

degradable materials it is essential to understand their fate in a waste stream. It is

2
this section of the “cradle to grave” life of a material that provided the thrust for

this work.

1.1 OBJECTIVE

The objective of this work was to understand qualitatively and quantitatively on a
laboratory scale the aerobic biodegradability of polymeric materials in the
presence of municipal sewage sludge. Biodegradation was studied in this work

from two perspectives - measurement and materials.

1.1.1 Biodegradation - Measurement perspective

There are various methods of measuring the biodegradation of polymeric
materials, some of which have been described in the next chapter along with their
advantages and disadvantages. In this work, the aerobic biodegradation of different
polymeric materials was measured using an inoculum derived from municipal
sewage sludge, based on ASTM-D5209 (several modifications were made to the
ASTM method to suit laboratory conditions). A theoretical analysis of the
biodegradation process inside this system was carried out. The effects of some of

the process parameters on biodegradation were also studied experimentally.

1.1.2 Biodegradation - Materials perspective

The inherent biodegradability of a material in a given environmental framework is
related to a number of factors most of which are characteristic of the material
itself. Some of these factors are its chemical structure, physical properties,
breakdown mechanisms and availability of specific enzyme-producing
microorganisms among others. In this work the inherent biodegradability of

3
different materials was studied and correlated to some of these factors. They
provided a better'lunderstanding in the design of materials intended to biodegrade

under environmental conditions.

Three categories were studied under Materials :-
1) A general category including some commercial polymers and other
materials of interest
2) Starch esters

3) Cellulose esters
1.2 SUMMARY OF THE CHAPTERS

This thesis is divided into nine chapters. Chapter 2 provides basic knowledge
about waste management infrastructure and biodegradable materials. Chapter 3
describes the different biodegradation measurement techniques in use, their
advantages and disadvantages, and a detailed description of the selected process
for the measurement of biodegradation. Chapter 4 gives a theoretical analysis of
the biodegradation process and an experimental analysis of the effects of some
parameters on the measurement process. Chapter 5 presents the results of
biodegradation tests canied out on some commercial polymers and other materials
of interest. Chapter 6 is an introduction to Chapters 7 and 8, and deals with the
structures and characterization techniques which are common to both the starches
and celluloses. Chapter 7 presents the effect of varying the extent of chemical
modification of starch on its biodegradation. Chapter 8 deals with cellulose acetate
having a high degree of substitution and presents the observed effect of

morphology on its biodegradation. A theoretical kinetic model for the aerobic

4
biodegradation of cellulose in this experimental system has also been proposed in
this chapter. Conclusions and recommendations for further work are presented in

Chapter 9.

Chapter 2

 

BIODEGRADABLE MATERIALS

 

2.1 WASTE MANAGEMENT INFRASTRUCTURE

Production of solid waste in the United States amounts to approximately 186,400
m. tons per day (68 Million m. tons per year) [EPA (1988)]. The Environmental
Protection Agency (EPA), has established a goal of a 75 volume percent reduction
in waste over the next eight years [Meister (1994)]. These reductions are supposed
to be achieved by a 25% reduction of waste generated at the source, a 25%
diversion of waste to recycling, and a 25% reduction of waste by incineration.
Degradable materials are one way of reducing waste volume at the source itself.
Paper, organic and food wastes comprise approximately 50% of landfill trash, all
of which are partially or fully biodegradable [EPA (1988)], (Figure 2.1). However,
approximately 20% of this waste mixture comprises of plastics, which are often,
and perhaps not always fairly, singled out as the major culprit in ultimate disposal

problems.
2.2 PLASTIC WASTE MANAGEMENT

Currently, most products are designed with little or no consideration for their
ultimate disposability or recyclability [Narayan, (1992)]. Options to manage our
plastic waste, are few and listed as follows [Narayan et al. (1992)]:

 

 

(lass
Nazis
(linr 2%

 

35% 20%

 

Figure 2.1 - Materials in MSW by volume
(Cubic yards)

 

7
a) Incineration - It has the benefit of regaining energy from waste, however, it is
capital intensive and there are questions about toxic emissions.
b) Recycling and source reduction- It is an accepted viable option, but only
where it is technically and economically feasible (i.e. where cleaning, reconverting
and reshipping costs do not exceed the virgin resin cost).
c) Landfills- They are a poor choice as a repository of plastic and organic waste
and are designed to retard biodegradation by providing little or no moisture with
negligible microbial activity.
(1) Composting- It is slowly gaining importance as the answer to the plastic waste

problem.

Plastics are formulated to be strong, light weight, durable and bioresistant
materials for various applications. They are resistant to biological degradation,
because,

a) microorganisms do not have polymer-specific enzymes capable of degrading
and utilizing most manmade polymers.

b) the hydrophobic character of plastics inhibit enzyme activity

0) the low surface area of plastics with their inherent high molecular weight
decreases rate of microbial activity.

The durability and indestructibility of plastics make them the materials of choice
for many applications, but this also creates problems when it enters the waste
stream. This leads us to the concept of designing and engineering new
biodegradable materials; materials that are plastics, i.e. strong, light-weight, easily
processed, energy efficient, excellent barrier properties, disposable (mainly for
reasons of hygiene and public health), yet break down under appropriate

environmental conditions just like it's organic counterpart. Clearly, not all plastics

8
can and should be made degradable. Single use disposable short-life packaging
materials, service ware items, and disposable non-wovens should be targets of new
material concepts that allow them to be fully compostable and to be incorporated

into the carbon cycle of the coo-system.
2.3 RATIONALE FOR BIODEGRADABLE POLYMERS

Plastics consist of carbon units assembled together to form complex polymeric
structures with desirable properties. These carbon units are derived from natural
resources and hence should be retumed to nature in order to complete nature's
own carbon cycle. By the law of conservation of mass we cannot make materials
disappear. However we can redistribute them in an ecologically sound manner by

nature's ecosystem [Narayan (1989), (1991 ), (1992)].

Figure 2.2 shows nature's carbon cycle and how biodegradable plastics can fit into
this cycle. As shown, green plants fix atmospheric carbon dioxide for their own
growth. These plants are then consumed by herbivores, which in turn are
consumed by carnivores, which in turn respire to form carbon dioxide and
ultimately dead organic matter. This organic matter is decomposed by
microorganisms in soil to form carbon dioxide and humic material. The humic
material is assimilated by plants and the humic carbon again gets into the carbon
cycle. Carbon-based polymers can also enter into this cycle if they can be
biodegraded by microorganisms found in waste streams and in the soil. Hence it
would be worthwhile to produce disposable plastics that can break down under
environmental conditions or in waste disposal systems into products that can be

assimilated by nature's ecosystem.

 

 

 

ATMOSPHERIC

 

 

C02

 

 

 

 

 

DEGRADABLE
PLASTIC

 

 

 

 

GREEN
PLANTS

 

 

 

 

HERBIVORES

 

 

 

 

 

 

 

 

 

CARNIVORES

 

 

SOIL LEVEL

 

 

 

 

 

DEAD ORGANIC MATTER

 

 

 

Decomposition

 

HUMUS

 

 

 

Decomposition

 

 

 

CARBON COMPOUNDS

 

+
C02

 

 

Figure 2.2 - Nature’s Carbon Cycle

 

 

10

2.4 BIODEGRADABLE PLASTICS - REGULATORY ACTIONS

As industry began implementing approaches to design environmentally benign
products, questions were raised about the practicality, efficacy, and the effects of
such products on the environment. This resulted in a number of regulatory actions.
A task force of several attorney generals issued recommendations (Green Report I
and II) on advertising related to environmentally friendly products [Webster
(1991)]. Green Report H states:

“It may be appropriate to make claims about the ‘biodegradability’ of a
product when that product is disposed of in a waste management facility
that is designed to take advantage of biodegradability and the product at
issue will safely break down at a sufficiently rapid rate and with enough
completeness when disposed of in that system to meet the standards set by

any existent state or federal regulations”

The US. Federal Trade Commission (FTC) guidelines [FTC (1992)] states:

“Unqualified degradability claims should be substantiated by evidence that
the product will completely break down and return to nature, that is
decompose into elements found in nature within a reasonably short period

,9

of time after consumers dispose of it in the customary way ............

2.5 BIODEGRADABLE MATERIALS IN THE INDUSTRY

Biodegradable materials found its way into the US. industry in the early nineties.
Starch-filled polyolefms with less than 10-15% starch were introduced as true

11

Table 2.1 - Current Status of the US Biodegradables industry

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

COMPANY BASE POLYMER FEEIETOCK COST, CAPACITY, MM
3% LII/YR
Cargfll, Minneapolis. MN Polylactide (EooPLA) Renewable Rm 1.00 3.00 10 (‘94 writeup): 250
Corn (mid-1996)
Eeodem. Wilmingtm. DE Polylactide oqrolyrners Renewable Resources. < 200 0.15 (‘94 scalwp)
Cheese M’ey, Can proj'd
Fidel. Atlanta GA Cellqrime. (Regenerated Renewable resources 2.15 1(1)
cellulose)
'Zerreea. (business unit (I 1C1 deOrydroxybutyrate-oo- Renewable resources - 8.00 - 0.66. additional
Anuims) hydroxyvalerae). (PHBV) aubohydrates (glucose). 10.0); 4.00 capacity slated ft! ‘96
orgmic acids proj'd is 11 - 22
Novmntn. Matedison. Italy Stardr-wrlhetic polymer blend Renewable resources + 1.60 - 250 50, in Ttnm'. Italy
curtailing agnox. 60% starch petrodremiml
Novm Prodms (Wrmer- 'Ihamqiastic stardr polymer Renewable resources, 2.00 - 3.00 1(1)
lambett). Morris Plains. NJ“ oorrpounded with 5-25% additives Stardt
Union Carbide. Darbury, CT Polyeaprolactme (TONE polymer) Petrodrenitnl 2.7 < 10
AirProdrdsJLOienimls. Polyvinyl alcohol (PVOH)& Petrodrenieal 1.0-1.25 150-200 (waersol
Allercown. PA 'Ihamoplam'c PVOH alloys (PVOH); PVOH ); 5 (VINEX)
(VINEX) 250—3 .00
(VINEX)
NatianlStard1&OrenlcaL Lowdsaarcheaer Renewableresomms. 2.00-3.00 Notavailable
Fridgeuata, NJ Stard‘i
UqumComStardrCo, Watawpenamtheunoplastic RenewableResouroes, 1.0-1.50 0.1(tilotsrale); 150
'g.MI modifiedstardres(AMYPOL) Starch slatedfaearly'96
Packag'ng Ted'nologes. Polyethylene oxide blertds Petrodnm'cal 3.00 10
Diego. CA (Elvirrplastic)
Rig-palm Co.. Ltd. omdensaion polyma' of glycols Parodenieal smut. 0.2 (pilot); 7(seni-
M'th alipimc dicarboxylic acitk 3.00 oorrmcial. end ‘94)
(BIONELLE)

 

 

12
biodegradable materials [Barenberg et al(l99l)], which created a setback to the
industry, since it is only the starch which undergoes biodegradation in these
materials. Since then, a lot of improvement has taken placed in designing
biodegradable materials with different material properties for different

applications. The current status of the US biodegradable industry is shown in
Table 2.1.

2.6 ENVIRONMENTALLY DEGRADABLE PLASTICS -
DEFINITIONS

The following definitions have been obtained from the proceedings of the ASTM
subcommittee on environmentally degradable plastics [ASTM, Toronto,(1989)].
They came up with two sets of definitions - one general and broad in scope and the

other more specific.

2.6.1 General Definitions

Degradable plastics- Plastic materials that disintegrate under environmental
conditions in a reasonable and demonstrable period of
time.

Biodegradable plastics- Plastic materials that disintegrate under environmental
conditions in a reasonable and demonstrable period of
time, where the primary mechanism is through the
action of microorganisms such as bacteria, yeast fungi

and algae.

13
Photodegradable plastics- Plastic materials that disintegrate under environmental
conditions in a reasonable and demonstrable period of

time, where the primary action is through the action of

sunlight.

2.6.2 Specific definitions

Degradable plastics- Plastic materials that undergo bond scission in the
backbone of a polymer through chemical, biological
and/or physical forces in the environment at a rate
which is reasonably accelerated, as compared to a
control, and which leads to fragmentation or
disintegration of the plastics.

Biodegradable plastics- Those degradable plastics where the primary
mechanism of degradation is through the action of
microorganisms such as bacteria, fungi, algae, yeast.

Photodegradable plastics- Those degradable plastics where the primary
mechanism of degradation is through the action of

sunlight

ISO 472:1988 (International Standards Organization) introduced a distinction
between degradation and deterioration as:

Degradation - A change in the chemical structure of a plastic involving a
deleterious change in properties.

Deterioration - A permanent change in the physical properties of a plastic

evidenced by impairment of these properties.

Chapter 3

 

MEASUREMENT OF BIODEGRADATION

 

Biodegradability of a material has become an increasingly desirable property for
plastic manufacturers due to plastic waste disposal and recycling problems. Hence,
the biodegradation potential of materials have been tested by various techniques
ever since interest in biodegradability was generated [Barenberg et al (1990)].
These methods ranged from relatively simple ones like soil burial tests to elaborate
ones like the ASTM standards D5338 and D5209. All of them, however give an
initial indication of the biodegradability of a material. Some of the measurement
techniques used have been discussed briefly here. The pros and cons of these

measurement techniques have also been discussed.

3.] GENERAL METHODS OF MEASUREMENT

3.1.1 FUNGAL TESTS

Plastic resins can be tested for degradation by fungi using specific growth
mediums. One such test is the ASTM D1924-63 in which a test specimen is placed
on or in a solid agar growth medium that is deficient in carbon. The medium and
specimens are inoculated with test microorganisms and incubated for three weeks.
Any growth that may occur is dependent on the utilization of a component of the
specimen as a carbon source by the organism. If a specimen shows heavy

microbial growth and concurrent loss of weight and mechanical properties, it is

14

15
considered evidence of biodegradability. Fungal tests have been carried out in the
past by various people [Union Carbide, Gilmore (1989), Graves (1994)]. This
method helps to determine the effect of specific microorganisms on the test
specimen, but does not quantify biodegradation directly. Also, it only accounts for
biodegradation in specific cultures and not in a mixed consortia of microorganisms

as in a waste environment.

3.1.2 SOIL BURIAL TESTS

This test consists of burying the plastic in moist soil for an extended period of time
ranging from 3 to 12 months [Breslin et al.(1993), Union Carbide, lannotti (1989),
Wool (1989)]. The plastic is in the form of tensile bars, films or other molded
articles. Mechanical property loss, weight loss, performance properties loss and
change in appearance (i.e., evidence of pitting or roughening of the surface
indicating bacterial growth), are measured periodically for the buried plastics. This
test method is not effective for tracking down end products and reaction

mechanisms owing to the diverse nature of the test matrix.

3.1.3 COMPOSTING EXPERIMENTS

In this test plastics are eprsed to a‘ simulated compost environment under
laboratory conditions. One such test, ASTM D5338 [ASTM D5338 (1992), Snook
(1994), Breslin (1993)] closely simulates, (but does not necessarily replicate) large
scale composting and is a good measure of the biodegradability of a material. The
test environment is a laboratory scale reactor which simulates a self-heating
composting system and which uses aeration to control maximum temperature.
Plastic exposure occurs in the presence of a synthetic waste undergoing aerobic

composting. The test materials are exposed to an inoculum of compost in two liter

16
vessels through which prehumidified, carbon dioxide free air is passed at a
constant flow rate. Carbon dioxide evolved as a result of aerobic biodegradation of
the composting samples is trapped in trapping solutions (BaOH or NaOH
solutions). The trapping solutions are removed and analyzed periodically for the
evolved carbon dioxide which is then correlated to percentage biodegradation of
the test material. A blank (compost with no test material) is also run in parallel
with the other test bottles to account for the carbon dioxide evolution from the
compost. The homogeneity of the compost is an important factor in these
experiments, and can lead to large standard deviations between replicates. Also,
the diverse nature of the test matrix makes it difficult to track down the end

products in the biodegradation of the test material.

3.1.4 ENZYMATIC ASSAYS

Enzyme assays have been canied out by various people in the past [Dijkistra
(1989), Angeline (1989), Williams (1989), Allenza (1989)]. In this method, the
plastic material is exposed to a specific enzyme(s) solution along with other
reactants. The rate and extent of biodegradation are measured by periodic testing
for the concentration of degradation products. As in the case of fungal tests, these
assays are more specific in their objective, i.e., the focus is degradation by specific.
enzymes rather than by environmental degradation in a mixed consortia of

microorganisms

3.1.5 LIQUID CULTURE EXPERIMENTS

All the biodegradability testing in this work has been done in liquid culture
experiments along the general lines of ASTM D5209, which is a standard test
method for determination of the aerobic biodegradability of polymeric materials in

l7
presence of municipal sewage sludge. This ASTM protocol was modified so as to
obtain optimum lbonditions for determination of the intrinsic biodegradability of
the test material. This test method consists of selection of plastic material for the
determination of aerobic biodegradability, obtaining activated sludge from a
municipal-waste water treatment plant and preparing inoculum, exposing the
plastic material along with nutrients to the aerated inoculum, measuring carbon
dioxide evolved as a function of time, and assessing the degree of
biodegradability. The percentage theoretical gas production based on measured or
calculated carbon content is reported with respect to time from which the degree of
biodegradability is assessed. A protein analysis can also be done on the residual
solution to complete the carbon balance for the polymer around the system. A

summary of the test method is shown in figure 3.1.

This test utilizes the polymeric carbon as the single carbon source, since the
carbon present in the inoculum is a small percentage (about 15%) of that in the
polymer. This allows for closer tracking of degradation products, and degradation
mechanism. There is also improved contact between the test material and the
microorganisms since the test solution is under constant agitation. The advantage
of this experiment over using an enzyme assay is that it comes closer to
biodegradation in the waste environment than pure enzyme cultures. The limitation
of this method is that it does not simulate the large scale composting system in
terms of temperatures and biodegradation rates in compost and waste piles. The
rest Of this chapter describes the selected method for the determination of aerobic
biodegradability.

18

 

Selection of plastic material for determination of aerobic
biodegradability

 

i

Obtaining activated sludge from a municipal waste water
treatment plant and preparing inoculum

 

i

[ Exposing plastic material to aerated inoculum ]

i

[ Measuring carbon dioxide evolved as a function of time 1

i

 

 

 

 

Assessing the degree of biodegradability based on the
percentage carbon conversion to CO,

 

Figure 3.1 - Summary of the test method

19
3.2 SELECTED MEASUREMENT PROCESS DESCRIPTION

3.2.1 SCOPE

This test method covered the determination of the degree and rate of aerobic
biodegradation of synthetic plastic materials (including formulation additives that
may be biodegradable) on exposure to activated-sewage sludge inoculum under
laboratory conditions. This test determined the intrinsic biodegradability of a

material and did not simulate large scale biodegradation in a compost environment.

3.2.2 INOCULUM TEST ORGANISMS

The test inoculum is prepared as specified by ASTM D5209. The source of test
organisms was activated sludge freshly sampled from the Waste Water Treatment
Plant of East Lansing, Michigan. It is a well-operated municipal sewage treatment
plant which receives minimal or no effluent from industry. This sludge is aerated
in the laboratory for 4 hours. About 500 ml of the mixed liquor is sampled and
homogenized for about 2 min at medium speed in a blender or equivalent high
speed mixer. The solution is then allowed to settle for about 30min. If the
supernatant still contains high levels of sludge suspended matter at the end of
30min, it is allowed to settle for another 30-40 min. Sufficient volume of the
supernatant is decanted to provide a 1-2% inoculum for each test Erlenmeyer
flask. Carry-over of sludge is avoided since it would interfere with the
measurement of CO; evolution during the test. Optionally, counts may be
performed on the supernatant fraction to determine microbial numbers. The
inoculum should contain 10‘5 to 206 colony forming units (CFU) per milliliter. It

should not be used on the day preparedand may be stored for two weeks at 4°C.

20

3.2.3 CHN ANALYSIS OF TEST MATERIAL

The test materialS' were weighed in a Perkin Elmer Autobalance Model AD-4 and
tested for their carbon content in the Perkin Elmer 2400 CHN Elemental Analyzer.
The PE 2400 CHN uses a combustion method to convert the sample elements to
simple gases (C02, H20 and N2). The sample is first oxidized in a pure oxygen
environment; the resulting gases are then controlled to exact conditions of
temperature, pressure and volume. Finally, the product gases are separated. Then,
under steady state conditions, the gases are measured as a function of thermal

conductivity.

Each sample’s CHN content was averaged over three runs, and the reproducibility

between the runs for C within 1% by wt.

3.2.4 REAGENTS AND TEST SOLUTION

Stock solutions were prepared and added to the experimental test flasks in the
following quantities [ASTM D5209]:-

5ml of ammonium sulfate solution (40g/L)

3ml of magnesium sulfate solution (22.5g/L)

3ml of calcium chloride solution (27.5g/L)

6ml of phosphate buffer (Potassium dihydrogen phosphate 8.5g/L, potassium
hydrogen phosphate 21.75g/L, sodium hydrogen phosphate 33.4g/L, ammonium
chloride 1.7g/L)

12ml of ferric chloride (0.25g/L)

The above stock solutions were added to the flasks along with 30ml of the liquid

inoculum. Water was added to the flasks and the solution level brought upto

21
1500ml. The polymer (test material) of known weight was added to the solution.
The carbon to nitrogen ratio maintained in the flask was less than 40:1, based on
the amounts of stock solutions used and the amount of carbon in the plastics,
which were adjusted accordingly. The test specimens were in the form of fine

powders or liquids.

3.2.5 EXPERIMENTAL SETUP

The schematic of the experimental setup is shown in Figure 3.2. It consists of the
carbon dioxide scrubbing apparatus, the carbon dioxide production, trapping and
measuring apparatus.

3.2.5.1 Carbon dioxide scrubbing and humidifying apparatus -

This apparatus consists of two 10L carboys, one containing about 5L of
concentrated NaOH solution and the other containing distilled water. The former
is used to scrub the incoming air free of carbon dioxide and the latter is used to
restore the air humidity to saturation level at the incubator room temperature. The
latter is incorporated to prevent evaporation of water in the flasks. The carboys
were connected in series, using vinyl tubing to a pressurized air system and the air
purged through the scrubbing solution and humidifier at a constant rate.

3.2.5.2 Carbon dioxide production and trapping apparatus-

This apparatus consists of 2L Erlenmeyer flasks with stoppers, each placed on a
magnetic stir plate. Each of these flasks were connected by means of vinyl tubing
at the inlet to an inlet air manifold. The outgoing air from the flasks was routed by
means of vinyl tubing to two bottles (or tubes) containing NaOH with 5%
sulfoorange as the carbondioxide trapping solution. The sulfo orange indicator

used has a color change pH range of 11.8 to 10.5.

mace-5.53m 25:5 23.: ..8 Baum .5553an . «am 0.53,.—

Ea:

   
      

     

  

Sci :3
2.2.9.! 52235:: Lona—9.0m «00

fl

 

 

xma: 32

 

 

 

 

 

 

 

 

A 22.5:
Essoa> Bo.— ..... _
.o o a
fine. .2
2829.30
_ _ __
2:393
2.2385:

mm

23

3.2.5.3 Experimental Procedure -

The apparatus is set up and the solutions prepared in the Erlenmeyer flasks as
described above. The Erlenmeyer flasks are first aerated with carbon dioxide free
air for 24 hr. to purge the system of carbondioxide. The test is started by bubbling
carbon dioxide free air through the solutions at the rate of 50-100ml per minute (1—
2 bubbles per sec) per Erlenmeyer flask. The air flow was maintained by a balance
between a pressurized air system connected to the inlet and a low vacuum at the
outlet thus preventing the build up of pressure inside the flasks. The air flow rate
was maintained at approximately the same rate in all the flasks by adjusting the
needle valves fixed in the stoppers of the flasks. The incoming air was scrubbed
and humidified prior to entering the flasks through a manifold. The outgoing air
was passed through the trapping solutions before going out through a manifold.
The carbon dioxide produced in each flask reacts with the trapping solution
producing carbonate. After a certain degree of carbon dioxide absorption, the
orange colored solution would turn yellow. The CO; trapping solutions would be
removed for measurement periodically and replaced by fresh trapping solutions.
Agitation is maintained throughout the test ensuring good mixing and contact. The
experiments were carried out in the incubator room where the temperature was
maintained at 28C. The initial test pH is 7+/-1. The pH was remeasured and
recorded at the end of the test.

The CO; filled trapping solutions were titrated in the Orion 960 Autochemistry
system with sulfuric acid and the result obtained in grams of carbon converted to
C02. Each sample was run in duplicate along with a blank. The CO; evolution was
calculated by subtracting the blank C02 values from the test flask C02 values, as

shown in figure 3.3.

24

C02 (blank) C02 (test flask)

 
 
   

 

 

Inoculum-1-
Inoculum + .
. Nutrients-I-
Nutnents .
test material

 

 

 

Blank Test flask

C02 (test material) = C02 (test flask) - C02 (blank)

Figure 3.3 - Computation of CO2 evolution from test flasks

25

3.2.6 MEASUREMENT OF ABSORBED CO;

The overall reaction taking place in the flask is:
C (test material) + 02 (air) = C0; + biomass
The reaction taking place during the carbon dioxide trapping is:

NaOH + co2 = NaHCO3. at high pH
NaOH + NaHCO3 = NazCO3 + H20 at lower pH

The titration curve [Snoeyink and Jenkins (1980)] for the carbonate system is

shown in Figure 3.4.

Total Alkalinity -

In the determination of the total alkalinity a known volume of the solution is
titrated with a standard solution solution of strong acid to a pH value in the
approximate range of 4 to 5. This endpoint is commonly indicated by the color
change of the indicator methyl orange; therefore the total alkalinity is often
referred to as the methyl orange alkalinity. The H+ added is the stoichiometric

amount required for the following reactions:

H” + OH’ = H20
W + HCO3- = H2C03
2H+ + C032. = H2CO3

The pH at the true endpoint of the total alkalinity titration should be that of a
solution of H2C03 and H20. This pH is referred to as pH(C02)

26

 

 
   
 

l

Sumo
acid
addition

Toni
alkalinity

Caustic

T alkalinity

 

 

  

 

--—__--_
I

 

 

 

 

 

 

Figure 3.4 - Titration Curve for the carbonate system. The trapping solutions
were titrated between pH8.3 and 4.5 with H2804 to calculate the amount of
CO; trapped.

Caustic and mrbonate Alkalinity -

For a carbonate system we can theoretically identify two more significant pH
values that occur during the course of an alkalinity titration. These are the
pH(HC03') and the pH(CO32').The value of pH(HC03') is about pH 8.3 and is
called the carbonate alkalinity, while p(HCO32‘) is generally between pH 10 and
11 and is called the caustic alkalinity. The former represents the pH of a solution
after H+ has been added to complete the two reactions:

27

H+ + 011.: H2O
H” + (2032' =HCO3'

The latter represents the pH of a solution after H+ has been added to complete only

the following reaction:

H+ + OH- = H2O

The carbonate alkalinity can be determined by the color change of the indicator
phenolphthalein and hence is often referred to as the phenolphthalein alkalinity.

If we make the approximation of complete reaction, then we can assume that by

pH 8.3 the reactions

OH - 'l' H+ = H20
and (:0,2 + H+ = Hcog'

are complete, and by about pH 4.3 to 4.7 the reaction

HCO3- + W = H2CO3
is complete. We can also deduce that each mole of C032' present will consume one
H+ when the solution is titrated to pH 8.3 and another H+ as it is titrated from pH

8.3 to 4.5. Thus, the volume of acid used for titration between pH 8.3 to 4.5 can be
used to calculate the total alkalinity of the system.

28

 

pause +9..
' _...__——slirrer

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

dispenser ——-———
dispenser -.._J;_;'_ ' "7'4:——stirrer
STACKED "“T". “' POW"
speed number anaIysis
tablet and
note card—7 '
SIIIIeI control cable cover
lfi - -- ——-» ' electrode
5 ...-..._ ._ . ' - \ tower
t__J ..--.._.._. -_ _.. a J
Fr.__—-—--—-—_______— I "A A f. ,. h
display C? C {3), E: X i.e. ,5 electrode
V V‘V‘ - hid tb
rj .. f: 1‘ /‘ r’f “ ’ ———eiectrode
V \. 3.2,- ~’ 4 .. .- holder
e
E .
3' Reseg'cr . .
' stirrer
g ,1' dispenser
5 E 10 probe
, i ' 15:

 

 

Figure 3.5 - The Orion 960 Autochemistry System

29

3.2.7 THE AUTOTITRATOR

The Orion 960 Autochemistry System was used to perform all the titrations in this
work. The front panel of the autotitrator is shown in figure 3.5. The Orion 960
Autochemistry System consists of the ORION 960 Module and an EA 940 pH/ISE
Meter which is placed on top of the module base [Instruction Manual]. The
module consists of a microprocessor, which controls the system, an autodispenser,
and an electrode tower. The EA 940 provides the display and the keyboard for the
entry of information. The technique used for the titration was endpoint analysis
based on a preset endpoint. This allowed the titration to proceed to a preset mV or

pH value. Titrations were performed as a sequence consisting of three steps.

Step1 - The titration was allowed to proceed to a pH of 12 - 10.5, depending on
the starting pH of the C02 filled trapping solution. This titration was a constant ml
titration in which standard increments of constant volume of titrant was added to
the sample to reach the end point.

Step2 - The titration was allowed to proceed to a pH of 8.3 in standard increments
of a constant mV. The increment size used ranged from 15 - 30mV depending on
the trapping solution.

Step 3 - The titration was allowed to proceed to pH of 4.5 in standard increments
of a constant mV. This increment size ranged from 5-15mV depending on the

concentration of the trapping solution.

The volume of titrant used for the titration in step 3 was used by the
microprocessor to compute the amount of carbon dioxide trapped in the trapping
solution and express as mg of carbon. The mV versus ml curve is shown in

Figure3.6.

30

 

mV

     

equivalence
point

I

 

 

1 I I I 1 _L l l 1

mL titrant

 

 

 

Figure 3.6 - Change in mV vs ml of titrant added for the autotitrator.

A small sample (3 1111-10 ml) of the trapping solution was diluted with water to
about 10-15ml and then subjected to titration. The titrant was sulfuric acid with a
normality approximately ten times that of the solution being titrated. Since the
levels of carbon dioxide evolution varied considerably from flask to flask, it was
often necessary to change the normality of the titrant, the amount of sample

dilution and the mV or ml step size used in the three steps.

3.2.8 CALCULATIONS

3.2.8.1 Theoretical CO; produced

The initial composition of the test material was determined by elemental analysis

using a CHN analyzer. This allowed the theoretical quantity of carbon dioxide

evolution to be calculated as illustrated in the following manner:

Material is w% carbon.

w/ 100 * mg of material charged = Y mg carbon charged to the Erlenmeyer flask.
C + 02 = C02

31

12 g C yield 44g C02
Y mg C yield 44/12 * Y mg CO;

3.2.8.2 Actual CO; evolved
The trapping solutions were titrated in two steps, upto pH 8.3 and from pH 8.3 to
pH 4.5. In some cases, where the starting pH was very high, the titration was done
in three steps, upto pH11.0, from pH11.0 to pH8.3, and from pH8.3 to pH4.5. The
volume of acid titrated between pH8.3 to pH4.5 was used by the autotitrator to
compute the C02 present in the trapping solution in the following manner:

Volume used for titration between pH 8.3 to pH 4.5=V1

Normality of titrant acid used = N1

Amount of dissolved C present in the trapping solution in grams
= V1 * N1 * 2(moles of HC03'/mole of acid) * 12(g of C/mole)

The CO; produced was computed from the above result.

3.2.8.3 Percentage C converted to CO;
Percentage Carbon converted to CO; was calculated as
= (mg C converted to C07] mg C present initially in the test material) * 100

Optionally, the protein content in the final solutions can be determined using

protein assay methods.

Chapter 4

 

THE BIODEGRADATION PROCESS

 

4.1 THE BASIC AEROBIC BIODEGRADATION EQUATION

The basic biodegradation equation for the aerobic biodegradation of any polymer

in the presence of a consortia of microorganisms can be generalized as:

Oxygen,
microorganisms microorganisms

 

 

Polymer > Monomer > C02 + humic mass
where, humic mass refers to all the material left behind after the evolution of C02.
This includes both the cell mass, the undegraded polymer and the smaller

monomer units formed during the breakdown process.

4.2 ANALYSIS OF THE BIODEGRADATION PROCESS

4.2.1 The Carbon balance
A material balance can be done for carbon around the aerobic biodegradation

system in the following manner:

Cpolymer = CC02 + CSOC + Cecil biomass + Cundegraded

where, Cpolym, = the amount of carbon originally present in the polymer (CHN
analysis)

32

33

Cco2 = the amount of carbon evolved as C02 during the testing
period
Csoc = the SOC (soluble organic carbon) present in solution at the

end of the testing period
Cundcgmded = the amount of carbon present in the residue as undegraded

material.

Cundcgmw can then calculated from the above equation to exactly determine the
extent of biodegradation during the testing period. Alternatively, the final solution
can be tested for the presence of the original polymeric material, and then tied in

with the above equation for verification.

4.2.2 Carbon mineralized to C02

The Ccoz is often used to determine the extent of biodegradation of the test
material. In this work, all the biodegradation data has been reported as the amount
of C mineralized to C02 and the percentage carbon converted to C02 based on
original C content. In some cases a protein analysis was done to obtain the C

converted to cell biomass.
4.3 FACTORS AFFECTING THE BIODEGRADATION PROCESS

4.3.1 EXPERIMENTAL FACTORS

a) Temperature

The temperature inside the incubator room was maintained at a constant value of
28°C throughout the test period at a constant value. Microorganisms have

different activities at different temperatures. Mesophilic bacteria act at lower

34
temperatures and thermophilic bacteria act at higher temperatures. In the case of
mixed substrates, as is the composting of mixed wastes, the entire consortia of
microorganisms in the compost matrix come into play for the breakdown of all the
different substrates present. However in the case of a single substrate, as is the
case with the liquid culture experiment, the microorganisms and it’s temperature
range of activity needed for the substrate breakdown is specific. Hence, the
composting experiments [ASTM D5338] are taken through a temperature cycle
during the course of the experiment, whereas, the liquid culture experiments are
carried out at a constant mesophilic temperature. At different temperatures within
the mesophilic range, there may be a difference in the rates and also extents of

biodegradation. This can be an area of consideration for further study.

b) Aeration rate

The aeration rate also affects the rate and extent of C02 production during the
aerobic biodegradation test in two ways. Firstly, the air provides the oxygen
necessary for the biodegradation process. Hence, insufficient amounts of oxygen
can become a limiting factor for the biodegradation equation, and thus govern the
levels of C02 produced. This would lead to an incorrect measure of the
biodegradability of a test material. Hence dissolved oxygen content is measured
and kept above the required levels, by maintaining the required air flow rate.
Secondly, the rate of air flow through the trapping solution affects the absorption
of CO; by the NaOH solution. A high flow rate would lead to a lower absorption,
whereas a lower flow rate would lead to relatively higher absorption. A balance
was struck between the two to satisfy both the requirements. The flow rate was

maintained at one to two bubbles per second through the trapping solution.

35

c) Agitation speed

The Speed of agitation of the contents of the experimental flasks also affects the
rate of biodegradation. The agitation provided serves to maintain good mixing and
contact between the microorganisms and the test material. If there was no mixing,
the active surface area of the test material for biodegradation would be greatly
decreased, thereby decreasing the rate of biodegradation. Thus diffusion could
become a limiting factor for the biodegradation equation instead of kinetics.
Hence, agitation levels were kept sufficiently high to ensure that the rates of
biodegradation were not diffusion limited. This factor was studied experimentally

to determine suitable agitation speeds required for the experiments.

4.3.2 MATERIAL PHYSICAL FACTORS

a) Morphology of the test material

The morphology of the test material affects the rate of biodegradation to a great
extent [Buchanan (1992)]. This is because the greater the surface area of the
material, the greater number of active sites are exposed to the microorganisms for
the breakdown mechanisms. The size of the pores, in case of porous materials,
also affects the rate of biodegradation. As is the case with the agitation speeds, the
surface morphology of the particles can thus limit the reaction to be mass transfer
limited or kinetic limited. Significant surface morphology effects were found

during the biodegradation studies on the cellulose acetates

b) Hydrophobicity of the test material
The hydrophobicity of the test material is known to affect it’s biodegradation
[Weirner (1991)]. If a material is soluble in water (the test medium), the

microorganisms have a medium to attach onto the active sites on the test material.

36
However, there have been some exceptions to this. PHB/V, a copolymer of
polyhydroxy butarate and valerate is a hydrophobic polymer, yet undergoes very

rapid biodegradation under experimental conditions.

c) Crystallinity of the test material

Biodegradation is also greatly affected by the crystallinity of a material [Weimer
(1991)]. Crystallinity decreases the biodegradability of a material. Native cellulose
is crystalline in nature and hence insoluble in water. When it is chemically
modified to a small extent, it’s crystallinity is decreased and it’s solubility in water

increases, and this makes it more biodegradable.

(1) Molecular Weight of the polymer

The molecular weight of the polymer is known to affect the rate of biodegradation
to some extent [Fqu et al(1986)]. Smaller chains take lesser time to be assimilated
by microorganisms than the longer chains. However, the overall extent of
biodegradation is not affected significantly for molecular weight changes in the
range of 50,00-100,000. This can also be an area for further study.

4.3.3 MICROBIOLOGICAL FACTORS
There are two factors which would affect the experimental data on the
biodegradation rates

a) Size of the microbial inoculum
The size of the inoculum indicates the number of colony forming units (CFU)
present in the microbial consortia. If there are less CFUs, it will limit the rate of

biodegradation. Hence, it is ensured that there are sufficient microorganisms for

37
the biodegradation of the entire polymer. As per ASTM D5209, a minimum of l -
20 * 106 CFUs/ml are specified for the given experiment.

b) Age of the microbial inoculum

Age of the microbial inoculum can affect the rate of biodegradation and the
reproducibility of replicates to an appreciable extent [Buchanan (1992)]. When the
sewage sludge inoculum is incubated with the test material for some time, it gets
acclimatized to the material. In microbiological terminology, the specific enzyme
producing microorganisms needed for the degradation of that particular material
flourish whereas the other microorganisms start to die. Thus, the resultant
inoculum after a certain period of time, is more concentrated in the favorable
microorganisms than the original inoculum. This increases the rate of
biodegradation to a considerable extent. Also, it increases the reproducibility
between the replicates in the experiment. ASTM D5209 prescribes a fresh
inoculum, which gives experimental deviations upto 110% . This inoculum was

used in all the experiments.

4.3.4 CHEMICAL COMPOSITION

If all the other conditions are satisfied, i.e., if the reaction is not diffusion limited,
oxygen limited and inoculum limited, then the chemical composition of the
polymer governs the rate, extent and mechanism of biodegradation. Hence, this is
the most important aspect which defines a polymer as biodegradable or not
biodegradable. Given the optimum conditions for biodegradation, a polymer can
biodegrade only if it’s chemical composition and structure is such that it can be
broken down and assimilated by microorganisms into smaller end products. This

factor has been studied in detail further on in this work.

38
4.4 LIMITING PARAMETERS IN THE BIODEGRADATION
EQUATION

The biodegradation process can be broken down into two basic steps:

4.4.1 Hydrolysis of the polymer to monomer by the hydrolytic enzyme

 

Polymer + tzO > Monomer
This step can be :

a) Diffusion limited

b) Inoculum limited

c) Substrate (polymer) limited

d) Kinetics limited
In this step, the attempt is made to optimize a) and b), so that the substrate is the
limiting reactant for the overall reaction and the rate equation is kinetic limited,
i.e., the substrate amount limits the total amount of C02 produced and the kinetics

of depolymerisation dictates the rate equation for this step.

4.4.2 Oxidation of monomer to CO;
Monomer + 02 > C02 + H20
This step can be:
a) Diffusion limited
b) Oxygen limited
c) substrate limited
(1) kinetics limited
Of the above mentioned parameters, a) and b) can be controlled, but c) and d)

 

follow after step 1. If the rate of step 1 is more than the rate of step 2, then the

39
monomer will start to accumulate and the step 2 reaction kinetics will govern the
production of C02. However if the rate of step 2 is more than the rate of step 1,

then the availability of the monomer will govern the rate of C02 production.

4.5 APPLICATION TO EXPERIMENTAL RATES OF POLYMER
SYSTEMS

For a polymer system, the above mentioned steps and mechanisms result in the
development of very complex equations as will be shown later on in the case of
cellulose and starch esters. This is because, usually there is more than one
hydrolytic enzyme acting during hydrolysis. In the case of starch and cellulose,
there are three to four enzymes acting synergistically at different rates to
accomplish the process of hydrolysis. When there are chemical modifications
involved, other mechanisms other than hydrolysis also come into picture, which
make the rate equation more complex. An attempt has been made to explain the
rate of CO; production mathematically in Chapter 8. Some of the parameters
involved in the biodegradation process were studied experimentally and are

reported in this chapter.

4.6 EFFECT OF AGITATION SPEEDS

The effect of agitation speed on the rate of biodegradation was studied using Poly
capro lactone (PCL) as the substrate.

4.6.1 EXPERIMENTALS

The CHN data on PCL as detennined by the Perkin Elmer 2400 Analyser was
%C= 63.3, %H = 8.85, %N = 0.087. Cellulose (microcrystalline avicel) was used

40
as the standard. A blank was also included in the set up which contained only the

nutrients and inoculum.

The amount of test material used in each flask was based on 0.8g of carbon and
were as follows:

PCL = 1.26 g, C content = 0.8 g

Cellulose = 2.00 g, C content = 0.8 g

The stock solutions used in each flask were as mentioned in chapter 3. 3 ml of
ammonium sulfate was added to the solution along with 30 ml of sewage sludge
inoculum. The amount of N in the sludge was assumed to be negligible and not
taken into account. Water was added to the flasks to bring up the level of solution
to 1500ml. The C/N ratio in the solution was calculated to be 25/ 1. PCL, cellulose
and the blank were all run in duplicate. Each flask was placed on a magnetic stir
plate which provided the agitation inside the flask. Three different speeds of
agitation were maintained They were speeds 3.0, 4.0, 5.0 on the stir plate which
corresponded to 700, 825, and 1000 rpm. One set was also run at zero speed.

Table 4.1 - Summary of biodegradation results

 

Agitation speed (rpm) % PCL-C to CO; (33 days)

 

 

 

 

0 14.10
700 30.89
825 32.71
1000 29.38

 

 

 

 

Cellulose, 825 rpm 75.06

 

41

 

 

 

 

 

 

BOT
e 701- l
v
‘6'
E -wt-Onnn
3 60-» '
a -€K-—700qnn
I; -4l-825qnn
- 50“-
fi —O—1000rpm
8 —D—cell,825rpm
3 40db
'u
a
t!
o
E :fllq- I o
8
L)
g. 20..
E
8
h
g 10.1

 

 

O . I I I I I I I
V V V V V U

5 10 15 20 25 30 35

 

Time in days

 

Figure 4.1 - Percentage conversion of PCL carbon to C02. Carbon

conversion with time at varying agitation rates was plotted for
PCLwhich was the substrate in all cases, except in the case of
cellulose which was included as a positive control. Each point is an
average of triplicate runs.

 

42

 

25.

 

Rate of C converted to C02 in mg/h
B

 

300

   
    
 

 

 

—O— Orpm
-O— 700rpm
+ 825rpm
—O— lOOOrpm
—D— cell,825rpm

-X—blank

 

 

 

 

15 20

Time in days

 

Figure 4.2 - Rate of PCL carbon converted to CO2 with time. The
rate of carbon conversion to CO; with time was plotted at different
speeds of agitation for PCL which was the substrate in all cases
except in the case of cellulose which was the positive control. The

rate of C conversion to CO, from the blank flask is also included.

 

 

43

 

501-

451-

400

% Cto C02

 

 

 

O a a a a
v T i f

0 200 400 600 800 1 000

Agitation speed (rpm)

 

 

 

Figure 4.3 - Relation between speed of agitation and
percentage C conversion to C02. Percentage of PCL C
converted to C02 in 33 days was plotted for different speeds

of agitation.

44

4.6.2 RESULTS AND DISCUSSION

A summary of mi: results of the above experiment is presented in Table 4.1. Figure
4.1 shows the rate of C converted to CO; with time and Figure 4.2 shows the
percentage C converted to CO; with time. It can be seen that the standard cellulose
reached a CO; evolution level of 70% within 30 days. PCL also started degrading
rapidly in the first few days and then slowed down to a steady rate after the first
week. The relationship between the speed of agitation and the percentage C
converted to C02 is shown in Figure 4.3. As can be seen the rate of C02
production was appreciably lower in the case of zero agitation as compared to the
other agitation speeds of 700, 825 and 1000 rpm. There was not much difference
between these three speeds in terms of C02 evolution. All the experiments in this
work were carried out at an agitation speed around 900rpm, thus ensuring that
there was adequate mixing between the test materials and the microorganisms and

that it was not diffusion limited.

4.7 EFFECT OF ACCLIMATISATION OF MICROBIAL
INOCULUM

An attempt was made to study the effect of acclimatisation of the microbial
inoculum on biodegradation. Three different samples were considered here, two

grades of cellulose acetate of DS 2.5, and a modified starch sample.

4.7.1 MATERIALS TESTED
The three samples were run in triplicate along with a blank and cellulose as the
standard. These materials were tested for their CHN contents by the CHN

analyser. All the samples were particulate solids between mesh size 80 and 140.

45
The materials tested were as follows :-
CA-398-3 (Eastman Kodak) - Cellulose Acetate DS 2.5, %C = 48.5, Mn = 30, 000
CA398-30 (Eastman Kodak) - Cellulose Acetate DS 2.5, %C = 48.5, M, = 50, 000
Modified starch (Michigan Biotechnology Institute) - %C = 49.1

4.7 .2 PREPARATION OF INOCULUM

Inoculum] - This inoculum was prepared as per the procedure mentioned in
Chapter 3 by obtaining activated sludge from the waste treatment plant. 50ml of
the inoculum was used in each flask for each of the three samples.

Inoculum2 - 1L of Inoculum l was centrifuged in an RC5C Sorvall refrigerated
Centrifuge and the resultant pellet suspended in 300 ml of water inside a 1L flask
along with 2.5 g of cellulose acetate DS 2.4, l g of cellulose and same amounts of
nutrient solutions used in the experimental flasks. The flask was stoppered and
incubated at 28°C on a shaker for 15 days. All agar medium was prepared in the
following manner -

a) 20 g of agar and 15 g of Nutrient Broth 2 were mixed with 1L of water in a 2L
flask.

b) The solution was autoclaved at 120°C for 20 mins.

c) The flask was taken out and allowed to cool slightly before pouring out into
petri-dishes.

About 1 ml of the incubated inoculum was plated on the agar medium in each
petridish and incubated at 30°C for 2 days. The colonies formed were then gently
scraped off the plates with the help of some water, and the total volume finally
made up to 150 ml.

46
Inoculum 3 - This inoculum was prepared in the same way as Inoculum 2 except
for cellulose acetate and cellulose being replaced by starch acetate and starch

respectively.

Each of the three samples were inoculated with Inoculum], the cellulose acetates
inoculated with Inoculum 2, and the modified starch inoculated with Inoculum3.

The C/N ratio maintained in the flask was approximately 25:1.

4.7.3 RESULTS AND DISCUSSION

No change was observed in the extent of biodegradation with acclimatisation. This
may be due to insufficient enrichment of the favorable microorganisms. However,
there was an appreciable decrease in the standard deviation between the replicates
with acclimatisation. Figures 4.4, 4.5 and 4.6 and Table 4.1 show the difference
between the normal inoculum and the acclirnatised inoculum with respect to the
standard deviation between replicates. This decrease in the standard deviation is

more prominent in the modified starch and the CA398-3O samples.

Table 4.2 - Standard deviations between replicates

 

 

 

 

 

 

 

 

Sample Normal inoculum Acclimatised inoculum
CA 398-3 1.62% 1.11%
CA398-30 9.54% 0.77%

Modified starch 11.37% 1.55%

 

This uniformity between replicates can be accounted for by the fact that during the
incubation of the original inoculum with the substrate, the growth of the substrate

47

 

20
18
16
14
12

Percentage C converted to C02
8

 

 

 

 

 

 

 

8 +CA 3
6
4
2
0 V V ; V I
O 10 20 30 40
Time in thys
N
O
U
8
c
if
0
>
5
U + CA-3 ac.
3“
§
'6
a.

 

 

 

 

 

Figure 4.4 - Effect of inoculum acclimatisation on CA-398-3. % C
conversion to CO2 was measured with time using CA39-3 as the
substrate for a fresh inoculum (top) and an inoculum that had
been acclimatised to the sample for 15 days (bottom). Each point
is an average of triplicates.

48

 

 

 

 

 

 

 

 

 

8 5° {—0—Mstachl
u 45
5 40
s 35
3 so
5 25
u 20
0
3° 15
e 1e
8
3 s
9" of : : : .
o 10 20 so 40
“meinthys
N
a 2
3 40
§ 35
a so
3 25
U 20
Si 15
g 10
§ 5
:‘1 o .
4o

 

 

 

 

Figure 4.5 - Effect of inoculum acclimatisation on Modified
starch. % C converted to C02 was measured with time
using modified starch as the substrate for a fresh inoculum
(top) and an inoculum that had been acclimatised to the
sample for 15 days (bottom). Each point is an average of
trilplicates.

49

 

 

 

 

 

 

 

 

 

 

 

8 50..
U
s 50“
“U
i”. 40‘
2
3 30 i +CA30
U
g 200
E
10eb
i
m o : V V ‘
O 10 20 3O 40
'Iimeindays
a e...
U
8 501»
I; 400
“=3
8 30 b l+CA-303C
U
3 201»
53 o : .L s :
O 10 20 30 4O
'Iimeinthys

 

 

 

Figure 4.6 - Effect of inoculum acclimatisation on CA 398-30.
% C converted to C02 with time was measured using CA398-
30 as the substrate for a fresh inoculum (top) and an inoculum
that had been acclimatised to the sample for 15 days (bottom).
Each point is an average of triplicate runs.

50
utilizing microorganisms was favored in comparison to the other microorganisms
present, which resulted in a more homogeneous culture for the replicates. Stepwise
enrichment of the inoculum to increase the population of the favorable

microorganisms is an area which should be explored in detail.

ll Chapter 5

BIODEGRADATION OF COMMERCIAL
POLYMERS

 

5.1 INTRODUCTION

Because of the growing awareness of environmental issues, most manufacturers of
plastics and other short term application materials are looking towards the final
fate of the plastics in the waste disposal streams. Increasingly, the need for
materials that are environmentally nonpersistent is being recognized as one
solution, particularly in those products which cannot be recycled. Most of the
common packaging plastics present in the current materials world possess very
little or relatively no biodegradability. However, there are some plastics which are
biodegradable but with poor mechanical properties. This is because,
hydrophobicity is a property of any material that is highly unfavorable from the
biodegradability standpoint but highly favorable from the mechanical property
standpoint. Biodegradability studies were carried out on some of the polymers

available commercially. The results are presented in this chapter.

5.2 POLYMERS TESTED

5.2.1 PHB/V‘ - PHIB/V, was obtained from Zeneca Bioproducts. Sold under the
trade name BIOPOL by Zeneca, it is a thermoplastic polyester produced from

51

52

agricultural feedstocks [Zeneca]. It is a copolymer of polyhydroxy butyrate and
valerate, currently produced with the valerate contents ranging from 5 to 20%,
either in powder or pellet form. This copolymer can be plasticized, filled,
nucleated, etc. much as conventional polymers. They also blend with polymers
containing some type of polar group such as PVC, chlorinated polyethylene,
polycarbonate and low melt nylons and alloys of these resins. They are not
compatible with nonpolar systems as represented by the polyolefins and
polystyrene[Galvin, T. J. (1989)]. The chemical structure of PHB/V is shown in
Figure 5.1.

 

 

 

 

 

 

 

 

N

R = n-alkyl pendant group of variable chain length
beta-hydroxybutyrate, R=methyl
beta-hydroxyvalerate, R=ethyl

Figure 5.1 - Structure of poly(beta-hydroxy alkanoate)

PHBN has been found to be biodegradable by the action of two extracellular

depolymerase enzymes; one enzyme predominantly cleaves dimer from the chain

while the other produces predominantly trimers [Gilmore (1990)].

53
5.2.2 Poly Caprolactone (PCL) - PCL was obtained from Union Carbide. It is a
homopolymer of caprolactone, a seven-membered ring compound. PCL resins are
miscible or mechanically compatible with many other plastics, like polyethylene,
polypropylene, PVC, PET etc. PCL has been shown to be biodegradable by

property and weight loss in soil burial tests [Union Carbide].

The polymer can be represented by the structure shown in Figure 5.2

0
||

HOR—O—I- C—GCH2)5-— O-I'fi’II

where, R = aliphatic segment
Figure 5.2 - Structure of PCL

Laboratory studies have been reported on the biodegradation pathway of PCL
which involves the formation of adipic acid, which undergoes fatty acid
degradation. In this process, two carbon atoms are successfully removed as acetyl
Coenzyme A, which enters the Citric Acid Cycle and becomes a source of energy

for the microorganism [Union Carbide].

5.2.3 Unmodified corn starch (Hylon 7) - This was obtained from National
Starch and Chemical Company. It is pure starch containing 70% amylose and 30%
arnylopectin, and has a molecular weight of around 10,000. It is insoluble in water.
The structure of starch is shown in Chapter 6. Starch is a naturally occurring

polysaccharide, known to be biodegradable in nature.

54
5.2.4 PCL/Starch blend - This material was an extruded 50/50 starch/PCL
polymer blend. The PCL and starch used were the ones described above. It was

prepared at the Composites Centre of Michigan State University.

5.2.5 Polyvinyl alcohol (PVOH) - This sample of PVOH was obtained from
Aldrich Chemicals. It was 99+ % hydrolyzed, molecular weight = 85,000 -
140,000. PVOH is a synthetic, water-soluble polymer, produced by the hydrolysis
of polyvinyl acetate, since vinyl alcohol exists only in it’s isomeric form,
acetaldehyde. Vinyl acetate is made by reacting acetic acid and ethylene. Varying
the molecular weight and percentage hydrolysis results in a broad range of

physical properties. The chemical structure of PVOH is shown in Figure5.3.

OH

Figure 5.3 - Chemical structure of Poly vinyl alcohol

The proposed mechanism for biodegradation in literature is initial oxidation of the
hydroxy] group by an oxidase to a ketone group followed by random cleavage of
the polymer chain at B-diketone formations by another hydrolase. This step is
followed by the endgroup hydrolysis to acetic acid, which finally produces carbon
dioxide and water [Philips (1991)]. However, it has also been reported that
increasing the percentage hydrolysis of PVOH decreases it’s biodegradation

considerably: ‘

55
5.2.6 Ethylene vinyl alcohol (EVOH) - This sample of EVOH was obtained from
EVAL Company of America, Grade LC-FlOlA. It is a copolymer of ethylene and
vinyl alcohol with 32% ethylene content, and a melting point of 183°C. It is used
for making blown films, melt phase forming and blow molding. it has a melt index

of 1.6 at 190C [EVAL (1992)].

5.2.7 Glycerol triacetate - This compound was obtained from Aldrich Chemicals.
It is a low molecular weight liquid used as a plasticizer. It has a melting point of

3°C. The structure of glycerol triacetate is shown in Figure 5.4.

cuzooc 0113

crrooccrr3

Figure 5.4 - Structure of Glycerol triacetate

5.2.8 Acrylic Coating - This material is sold under the trade name “AquaCoat
584F” by International Blending Corporation (IBC), Minneapolis. It is a thick
viscous liquid with a recommended viscosity of 28 to 35 seconds through a
number 3 Zahn cup. It is used as a coating material for various paper products and

other applications.

5.2.9 9-11 Acid - This product was obtained from Cas Chem and is a derivative of
Castor oil. Conversion of dehydrated castor oil by hydrolysis to acid and

subsequent distillation yields 9-11 acid, a high purity conjugated fatty acid. In 9-

56
11 acid, the total linoleic content is 94%, and combined oleic and saturated acids
content is less than 10%. This product has properties that can be utilized in a broad
list of resin/polymer systems generally directed to coatings, inks and adhesives.
They are applicable to resin systems based on 100% solids, solvent based and
water based formulations [Cas Chem (1992)]. It is insoluble in water, but soluble

in most organic solvents.
5.3 EXPERIMENTALS

The CHN data of the materials tested are given below

Glycerol triacetate - %C = 49.57, %H = 6.13, %N = 0.15

PHB/V powder - %C = 56.79, %H = 7.22, %N = 0.21

PCL powder - %C = 63.3, %H = 8.85, %N = 0.087 _
Unmodified corn starch (Hylon7) - %C =43.26, %H = 6.52, %N = 0.146
PCL/starch blend - %C = 50.

PVOH - %C = 52.73, %H = 8.79, %N = 0.077

EVOH - %C = 60.45, %H = 10.27, %N = 0.063

AquaCoat 584F - %C = 46.3, %H = 8.59, %N = 1.56

Cellulose was used as the standard in all cases.

5.3.1 Inoculums and test solutions
These materials were run in different sets of experiments conducted in different
time frames. The sample concentrations used in the different experimental setups

were as follows:-

57
Experimental Setup #1 -
Aqua Coat 584F = 2.5g, C content = 1.16g
9-1l acid = 2.0g, C content = 1.6g
Cellulose = 4.0g, C content = 1.6g
The C/N ratio in this setup was approximately 35/1, and for the AquaCoat 26/1.

Experimental Setup #2 -
Glycerol triacetate = 3.23g, C content = 1.6g
Cellulose as standard = 4.00g, C content = 1.6g

The C/N ratio in this setup was’approximately 35/1.

Experimental Setup #3 -

PHB/V= 2.64g, C content = 1.5g

PCL = 2.36g, C content = 1.5g

Unmodified starch = 3.47g, C content = 1.5g
PVOH = 2.85g, C content = 1.5g

EVOH = 2.48g, C content = 1.5g

Cellulose as stande = 3.76g, C content = 1.5g.
The C/N ratio was calculated to be 33/ 1.

Experimental setup #4 -

PCL/starch blend = 1.6g, C content = 0.8g
Cellulose as standard = 2.0g, C content = 0.8g
The C/N ratio was calculated to be 25/1.

58

 

 

100‘

Percentage C converted to C02

 

 

 

Time in days

 

Figure 5.5 - Percentage C converted to CO, with time for
AquaCoat584F and 9-llacid. Cellulose was used as the positive
control. Each line represents an average of duplicate runs. Blank
values have not been subtracted in this set of results.

 

59

 

 

    

1.0 '
N 0.8 'l
O
U .
2
g 0.6 '
I:
0
>
8
° 0.4 ‘
U llkmmm:
° ° Glycerol Triacetate
l:
.3 0.2 d
U
i!
la.

 

 

0 10 20 30
Time (Days) ,

 

Figure 5.6- Percentage initial C converted to CO2 with time
for Glycerol triacetate. Cellulose was used as the positive
control. Each line represents an average of duplicate runs.

 

60

 

 

70.00 -
l

+ Cellulo
60.00 c v-

/ + HYIOII 7
50.00 - - _ / + PHB/V

4o.oo-- ,~ "x‘ PCL

/ / —e— PVOH

 

 

 

Percentage C converted to C02

10.00J

l.
i

 

 

0.00 + .
0.00 10.00 20.00 30.00 40.00 50.00

Timindays

 

Figure 5.7 - Percentage of initial polymeric C converted to CO,
with time for various commercial polymers. Cellulose was used as
the positive control. Cellulose, Starch, PCL and PHB/V represent
the average of triplicate runs; PVOH and EVOH represent the
average of duplicate runs.

 

61

 

 

Rate of C converted to C02 in mg/daj

60.00 -

50.00 '

V

40.00 -

V

20.00 s

V

10.00 ‘

 

 

 

 

+ Cellulose

—I— Hylon 7

—X- PCL

-D— EVOH

 

 

X

O.m 1‘ : : I '1‘”

Time in days

or) 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

 

Figure 5.8 - Rate of initial polymeric C conversion to C02

with time for various commercial polymers.

 

 

62

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

70 a -
N V
8 —O-— Cellulose
3 + PCL/Starch
1:
8
'6
>
i:
8
U
i“
5
§
0
a.
50
40 a p
a?
a
h
E.
N
O
U
8
g + Cellulose
g +PCUStarch
E
8
U
“5 I
8
a
a 9 r.
50
Time in thys

 

 

 

Figure 5.9 - Initial polymeric carbon converted to CO2 with time
for the 50/50 PCL/starch blend. Cellulose is included as the
positive control. Each point is an average of duplicate runs.

63

 

 

 

 

 

 

 

 

 

-O—Cellulose
80.00 --
+Hylon 7

70.00 -- + PHB/V
N
8 —x- PCL
e 60.00 --
“ -O— PVOH
'8
E 50.00 —+- EVOH
>
=
8 4000
U
3" 30 oo
3 e
i
'5 20.00 ..
A:

10.00 «-

0.00 . . . .

0.00 10.00 20.00 30.00 40.00 50.00

 

Figure 5.10 - Percentage of initial polymeric C converted to CO,
with time for various commercial polymers (no blank correction).
This graph is a replicate of Figure 5.7 except that no blank
correction has been applied here.

 

 

 

 

 

 

 

 

 

 

 

60.00 -- ° Elanl:
-O—Celuiose
+ Hylon 7
in
g 5000 . -x— Pl-B/V
E: -x— PCL
.5 -+-- PVOH
N 40.00 "' —— EVOH
O
U
G
d
B 30.00 ..
t
a)
>
=
8 20.00 ..
U
a.
O
N I» , _
m 10.00 < -
_1L_ ——fi
Cam 1’ V' T 4V
0.00 10.00 20 00 30.00 40 00 50 00

Tine in days

 

Figure 5.11- Rate of initial polymeric C converted to CO; with
time for various commercial polymers (no blank correction). This
graph is a replicate of Figure 5.8 except that no blank correction
has been applied.

 

65

 

 

+ Cellulose
+ PCL/starch

451-

I.
*

 

 

 

 

 

 

Rate of C converted to C02 in mg/day

 

50

 

 

 

 

—I- Cellulose
-a— PCL/Starch

 

 

 

Percentage C converted to C02

 

 

 

Tine in days

 

Figure 5.12 - Initial polymeric carbon converted to C02 with
time for the 50/50 PCL/starch blend (no blank correction).
This graph is a replicate of Figure 5.9 except that no blank
correction has been applied here.

 

66

5.3.2 RESULTS AND DISCUSSION

Figures 5.5 through 5.9 Show the %C converted to C02 and rate of C02 produced
with time for all materials tested. Figures 5.10 through 5.12 Show the results of
Experimental setups #3 and #4 without the blank correction. Table 5.1 gives a
summary of the biodegradation results as well as the standard deviations between

replicates.

Table 5.1 - Summary of biodegradation results for all setups

 

 

 

 

 

 

Material %C to CO; St. deviation
Setup #1 Cellulose 77.8‘ -
38 days Aqua Coat 584F 15.0’ -
9-11 acid 511‘ -
Setup#2 Cellulose 70‘ -
30 days Glycerol triacetate 78" -
Cellulose 60.14 7.3%
PHB/V 62.14 6.5%
Setup #3 PCL 31.26 4.1%
39 days Starch 50.17 7.1%
PVOH 6.19 0.9%
EVOH 15.61 9.9%
Setup #4 Cellulose 64.55 4.2%
44 days PCL/Starch blend 38.69 5.6%

 

 

 

 

* Blank values not subtracted

 

67
The first and second experimental setups were conducted during the early part of
this work. In these two setups, each tube containing the trapping solutions were
removed only when the solution turned yellow. This resulted in more data points
for the faster degrading materials than the slower ones. Hence, the CO; evolution
curves have been obtained by curvefitting data points as shown. Also, this method
made it difficult to subtract the blank values from the test flask values.

AquaCoat 584F showed a very low rate and extent of biodegradation. This is
important because the biodegradability of the coating affects the biodegradability
of coated paperboard and other coated articles by creating a non permeable barrier
to the degrading microorganisms. Cellulose, the positive standard reached a C02
evolution of 78% in 38 days. The 9-11 acid also showed substantial
biodegradation within 38 days showing it’s potential for further biodegradation. In
the second setup, Glycerol triacetate underwent rapid degradation to C02 within
the first few days. Cellulose reached a C02 evolution of 70% in 30 days.

In all the remaining setups, the trapping solutions were removed for titration
periodically, all at the same time, irrespective of the levels of C02 generation. This
allowed for averaging of the duplicate or triplicate runs, subtracting blank values
and for calculating the standard deviations between the replicates. PHB/V
underwent rapid biodegradation within the first few days at the same rate as
cellulose. PCL on the other hand had a slower, but almost constant rate throughout
the time frame of the experiment. Starch also underwent rapid biodegradation to
about 50% of C02 evolution within thirty days. PVOH was recalcitrant to
biodegradation throughout the test period. It is interesting to note that EVOH
showed a slightly higher percentage biodegradation than PVOH (although it also

68

had a higher standard deviation). The biodegradability of PVOH, well established
in literature, is'hlso very sensitive to the percent hydrolysis. Increasing the percent
hydrolysis of PVOH increases it’s crystallinity, and reduces it’s solubility [Philips,
J .,(199l)]. Crystallinity affects biodegradability adversely. The sample of PVOH
used in this case, was 99+% hydrolyzed, which accounts for the low extent of
biodegradation achieved in this experiment. EVOH, on the other hand was a
copolymer of PVOH and ethylene, where the latter has been established as highly
recalcitrant to biodegradation in literature. However, the presence of ethylene in
the copolymer decreases it’s crystallinity, thus rendering the vinyl alcohol
segments in the chain to be more susceptible to enzymatic attack. In the last setup,
the PCL/starch blend showed a steady biodegradation rate throughout the time
frame of the experiment. In this case, since the blend was in the form of small
extruded pieces, the surface area of the pieces might have dictated the rate of
biodegradation.

Chapter 6

 

CELLULOSE AND STARCH ESTERS

 

6.1 BACKGROUND

The complex structure of cellulose and cellulosic materials introduces
considerable problems to a researcher attempting to measure it’s biodegradation.
The rate and extent of cellulose biodegradation by microorganisms and their
enzymes is dependent in part on various physical and chemical parameters.
Moreover, because cellulose is a substrate of complex structure, a number of
substrate related factors are major determinants of biodegradability [Weimer
( 1991)]. Two general types of substrates have been used to measure cellulose
biodegradation. The first group includes relatively unaltered natural substrates
such as pure crystalline cellulose; the second includes modified cellulose
substrates whose biodegradation occurs more rapidly (e.g., substituted celluloses)

or is more easily observed (e.g., dyed celluloses).

Cellulose derivatives have proved to be more complicated in it’s kinetics of
biodegradation than unmodified cellulose. Considerable work has been done on
cellulose derivatives like cellulose acetate and carboxy methyl cellulose. However,
most of the work so far done in this field has been focused on derivatives with a

low degree of substitution of the chemically modifying group; only recently has

69

70
some detailed work been done on derivatives with a higher degree of substitution

of the modifying group.

Similar work has been done on starch and starch derivatives, though to a lesser
extent. There are some basic differences between the structure of starch and
cellulose, but their behavior in a consortia of microorganisms is essentially the
same. In this work, cellulose and starch esters were studied in detail, the results of

which have been reported in the next two chapters.

In order to study the effects of chemical or physical modifications on the
biodegradation of these esters, it is essential to characterize them properly. Some
fundamental points and characterization techniques which were common to both

the esters have been discussed in this chapter.
6.2 THEORETICAL

6.2.1 STRUCTURE OF STARCH

Starch is the reserve food in plants, and it’s granules are large enough to be seen in
many plant cells when examined through a microscope [Bailey and Ollis (1986)].
Starch consists of two fractions: amylose and amylopectin. Both are
homopolymers of -D- glucopyranosyl units, except that amylose is a linear

polymer and amylopectin is a branched polymer.

Amylose - Amylose is the straight chain polymer of glucose subunits with

molecular weight ranging from several thousand to half a million. Amylose

71
typically comprises about 20 percent of starch. It‘s chains are formed by the

presence of or -l,4-glycosidic bonds as shown in the Figure 6.].

OH OH

I |

114—”\1‘ r71:— ”\1'

_I\j" f/I_,_J\C" I/l_0_
I I I

11 OH . H OH

 

 

Figure 6.1 - Structure of unmodified starch (Amylose fraction)

Amylopectin - While the amylose fraction of starch consists of straight chain,
water-insoluble polymers, the bulk of starch is amylopectin. The structure of

amylopectin is shown in Figure 6.2.

mutanéx

Figure 6.2- Structure of unmodified starch (Amylopectin)

72
Amylopectin is distinguished by a substantial amount of branching. Branches
occur from the ends of the amylose segments averaging 25 glucose units in length.
Such structures arise when condensation occurs between the glycosidic -OH on
one chain and the 6 carbon on another glucose. The Amylopectin is formed by

crosslinking between the linear chains by the 1-6 linkages.

6.2.2 STRUCTURE OF CELLULOSE

Cellulose, a major structural component of all plant cells is the most abundant
organic compound on earth. Although the glycosidic linkage in cellulose occurs
between the 1 and 4 carbons of successive glucose units, the subunits are bonded

differently than in starch. The structure of cellulose is shown in figure 6.3.

l" i"
T/lC—_ 0W V:— \p
_l\‘l" l/ T—N’ ‘7

II OH

 

lH (|)H

Figure 6.3 - Structure of cellulose

The difference in structure between starch and cellulose is significant. While many

microorganisms, plants and animals possess the enzymes necessary to break down

the [31,4- glycosidic bonds of starch, very few living creatures can hydrolyze the

73
[31,4 bonds of cellulose [Bailey and Ollis(l986)]. One of the common products of
enzymatic cellulose hydrolysis is cellobiose, a dimer of two glucose units joined

by a B-1,4-glycosidic linkage.

6.2.3 STRUCTURE OF CELLULOSE AND STARCH ESTERS

The cellulose and starch esters are produced by esterification. There are three OH
groups on the monomer unit . They are at the C2, C3, and C6 positions as shown
in Figure 6.4. Out of these three the C6-OH is the most reactive OH group, and the
C2 and C3-OHs are less reactive. Hence during a substitution reaction, the C6-OH
is first substituted, and then the C2 and C3-OHs. The three OH groups on a

monomer unit of the polysaccharide are shown in Figure 6.4.

OH on
Figure 6.4- Position of OH groups on the monomer unit of cellulose/starch.
The substitution of the acetyl groups on the monomer unit results in the structure

shown in Figure 6.5 . This figure shows substitution at all three OH groups of the

monomer unit.

74
1:60: OCCH3

oioccn3 oioccng,

Figure 6.5 - Structure of a triacetate monomer unit of cellulose or starch

6.3 EXPERIMENTAL CHARACTERIZATION OF CELLULOSE
AND STARCH ESTERS

Characterization of the esters was done based on two factors

a) Degree of substitution

b) Position of substitution

The former was done with the help of 1H NMR and the latter with the help of 13C
NMR.

6.3.1 DEGREE OF SUBSTITUTION

The properties of cellulose and starch acetates (propionates, etc.) are a function of
the acetyl content, the type of starch, the size and shape of it’s molecular
components, and the method of pretreatment. Measurement of the acetyl content is

a prime method for characterizing these acetates [Whistler (1972)].

75
Acetyl content

The percentage acetyl content of the acetate is defined as

(CH3 c0)!“W * DS

%Acteyl = * 100
(Acetate)mw

 

In the above expression,
DS = degree of substitution of the acetate.
(CH3CO)m.w.= 43
(acetate)m.w. = (CH7Os)m.w. + (H3 - DS)m.w. + ((CH3CO)m.w * DS)
=159+3-DS+43DS
= 162 + 42 DS

Degree of substitution
The definition of the DS of the acetate follows from the above expression and is

given as;

162 * % Acetyl

 

Degree of Sustitution ( D.S ) —
4300 - ( 42 * % Acetyl)

The DS is the number of OH groups out of the three OH groups on a monomer
unit that is substituted by the acetyl group.
In the case of propionate, the (CH3CO)mw is replaced by (CH3CH2CO)mw = 57

and the calculations carried out as before.

6.3.1.1 DETERMINATION OF DS BY THE METHOD OF TITRATION
The D8 of cellulose and starch acetates is usually determined by the conventional
titration method [Whistler (1972)]. The sample is ground, dried for 2hr at 105 +/-

76
3°C and cooled in a desicator. lg of the sample is transferred to a 250 ml. flask,
and 50ml. of a 75% ethanol solution in distilled water is added. The loosely
stoppered flask is agitated, warmed to 50°C, held at that temperature for 30mins.,
and cooled; 40ml of 0.5N NaOH is added while swirling. The flask is stoppered,
allowed to stand for 72hr. with occasional stirring. The excess alkali is back
titrated with standard 0.5N HCl using phenolphthalein as indicator. The solution
is allowed to stand for 2hr., so that any additional alkali leaching from the solution
can be titrated. A blank is titrated at the same time, substituting the original

starch/cellulose for the sample. The % acetyl is calculated as shown

[ml.(Blank) - ml.(Sample)] * normality of the acid * 0.043 * 100

 

% Aw” = Sample wt., g. (thy basis)

The titration method yields experimental error which results in appreciable
deviations between replicates. It is also a time consuming method. A new method
was evolved in this work which determined the DS by NMR. This method reduced
experimental errors to a great extent and also saved a lot of time and effort. This

method has been described in detail.

6.3.1.2 DETERMINATION OF DS BY PROTON NMR

Nuclear Magnetic Resonance is a highly dependable and less time consuming
process as compared to the traditional titration method, and is a viable alternative
for the determination of the DS of these esters. The NMR method has been used
by Buchanan et. al. [Buchanan (1992)] for the determination of the DS of cellulose

acetates with excellent accuracy. However, it has been used very little for the

77
starch esters. NMR was used to determine all the DS values used in this work for

both the starch and cellulose esters.

Sample preparation

a) About 50-100 mg of the modified starch or cellulose was weighed accurately in
a small sample bottle. DMSOd6 absorbs moisture rapidly, hence care was taken to
avoid exposure to atmospheric air during the sample preparation.

b) About 1-1.5 ml of DMSOd6 was added to the sample with the help of a pipette.
The bottle was covered with a cap and placed on a stir cum hot plate. The stirrer
was turned on and the temperature raised to 70-80°C, so that the sample dissolved
quickly (the modified starches are not readily soluble in DMSOd6, and heating the
solution decreases the dissolving time). In order to prevent moisture from
accumulating in the bottle, the air inside the bottle was flushed with nitrogen
during the heating process.

c) After half an hour (or until the solution became clear), the heat was turned off.
1-2 drops of TFA/DMSOd6 (50/50 vol./vol. mixture) was added to the solution
very carefully with the help of a pipette. The solution was allowed to stir for a few
seconds more and then taken off the stirrer.

d) The solution was filtered and transferred from the bottle into a clean NMR tube
with the help of a pipette and some quartz wool. (This is essential in the case of
the starch acetates and propionates, since the higher DS starch acetates are not
very soluble and hence remain as small particles in the solution which spoils the

spectrum). The height of the solution in the NMR tube was about 5 cm.

78

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 6.7 - Proton NMR of Starch propionate without TFA

_T—r ' l ' I I ' ' ‘ I ' ' ' ' l ' ' ' ‘ l ' ' ' ' l l [I I l I ' '
6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppfl
Figure 6.6 - Proton NMR of Cellulose acetate without TFA
' I I I I ' ' I I r1 j I I T I T r ' ' T ' ] ' I ' ' I I V I V I I r r r I r r r r I r r r r I rfi r r I r r r r I
6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

79

 

 

 

 

Figure 6.8 - Proton NMR of CA with TFA

/.

/l
/ .
/ /\/LJ
1 I T f V I ‘ T V V T 1‘1 V V T 1 I V 1 v I V V V V 17 T V f V
B 5 A 3 2 mm

Figure 6.9 - Proton NMR of Starch propionate with TFA

 

 

 

\“

 

 

D.

80

Proton NMR spectra

In order to calculate the D8 of these esters, it is essential to get a good spectrum.
During the early part of this work, TFA was not used, hence some problems were
encountered due to the presence of water peaks. The gradual improvements in the
spectra with a few modifications are presented here. DMSOd6 absorbs moisture
rapidly, hence care was taken to avoid exposure to atmospheric air during the
sample preparation. Cellulose acetate of known DS was used as a standard to
determine the accuracy of this method. The proton NMR spectrum of cellulose
acetate DS 2.5 without TFA in Figure 6.6 shows the methyl peak and the ring
protons. As can be seen, there is a very tall peak in the region of the ring protons.
The spectrum of starch propionate without TFA in Figure 6.7 shows the methyl
and methylene peaks and the ring protons. Here also there is very tall peak in the
region of the ring protons. This tall peak is due to the water impurity in the sample
which makes it impossible to compute the correct peak area of the ring protons.
This problem was taken care of by adding 1-2 drops of TFA (Tri-flouro-acetic
acid) [Buchanan (1992)] which removed the water hydroxyls and the ring
hydroxyls from the region of interest. The resultant spectra were as shown in

Figures 6.8 and 6.9.

Initial spectra obtained at room temperature did not provide very good shirnming
in the instrument. This was due to the viscosity of the sample which was high
owing to high sample concentrations in the solution. Hence, the spectra were
obtained at 60-80C to get better shimming. Phasing of the spectra was very
important in the integrating the peak areas for accurate determination of the US of
the sample. Inaccurately phased spectrums can give inaccurate peak areas and DS

values.

81

Calculation of DS from proton NMR

The chemical stru'bture of starch is as shown in Figure 6.1. There are 5 protons and
2 hydroxyl groups in the ring, two methylene protons and one hydroxyl on the
sixth carbon. The ring protons, methylene protons of C6 and the hydroxyls are
spread out between 2.8 and 5.5 ppm approximately, whereas the acid group
protons are at 2.0 or 1.0 ppm depending upon the acid group, i.e., acetic or
propionic. When TFA is added to the sample, it removes the hydroxyls of the ring
away from the ring proton region. Thus the area under the peaks occurring
between 2.8 and 5.6 ppm are due to 5 ring protons and 2 C6 protons. The acid
methyl peak is due to 3*DS number of protons.

Thus,

Area under the acid methyl peak 3 * DS

 

 

Area under the ring and methylene protons peak 7

The DS was thus calculated by integrating the peak areas.

6.3.2 POSITION OF SUBSTITUTION

The carbon atoms present in the acetate and propionates and their positions within
the molecule with respect to other atoms was investigated by C13 NMR. An
attempt was made to determine the positional substitution of the acetyl groups, i.e.,
whether they are substituted in the C2-OH, C3-OH or C6-OH positions. All the
. C13 NMR was canied out on the Varian 500mHz instrument using a 10mm.
probe. The concentrations used were in the range of 8-10% wt/vol. (IOOmg/ml
approx.) of the sample. The solvent used was Di-methyl-sulfoxide-d6 (DMSOd6).

82

 

 

 

 

 

Ina—en‘sJenna‘s-amneuh—h‘Anluu-LJr—LLufluawdfi- ulna.- J a- . .. . i. . undue...” n... ._u..‘-

220 200 180 150 140 120 100 8’.“ 60 40 20 00111

 

 

 

 

 

 

 

VIIITIIIIIII.IIIIIIT7ITTIIIFIIITfiliIIIITIiII

100 95 93 85 80 75 7O 65 60

Figure 6.10 - C13 NMR of unmodified Starch

83

 

 

 

 

 

Imus—J 1.1ng

I—IIIIIIIrIIIIIIIIIIIIIIIIIIIIYIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII‘IIIIIIIIIIIIII[IIIIIIIIIIIIII‘IIIII

200 180 160 140 120 100 80 -60 40 20 Don
N
8
a
is}

 

 

 

 

 

TWIIIIIYIITIT]IIIIIIIIIIIITTIIIII‘IIIIIIIIIIIIIIITTIIII

177 176 175 174 173 172 171 170 169 168 00m

Figure 6.11 - C13 NMR of starch propionate of DS 3.0

84

 

 

 

 

 

 

 

1 . I t-..i. A...J.... “mamL-U‘al.

mammal-sac mammalian . “Hate.
100 80 60 40 20 0 00m

. La ram¢uuw

200 180 160 140 120

 

 

 

WU. NMC‘N‘M‘WN-WAAIJ.‘ \ ‘M 1,..‘1‘ {m 1 ‘\ w ; ‘15-‘50“ . \‘v I“? “K, “)5“va hN-I”,

TTLI[IIIIIITITITIIIIIIII1VIII—[ITITIITIIIII’IIl—III

175 174 173 172 171 170 169 166 167 166 165 164 com

Figure 6.12 - C13 NMR of starch acetate DS2.0

 

85
Figure 6.10 shows the C13 NMR of unmodified starch. The 6 peaks represent the
6 carbon atoms in the starch structure. Based on their neighboring atoms, each of
these peaks were identified as C1, C4, C5, C2, C3 and C6 from left to right of the
spectrum. C13 NMR cannot be used for quantitative analysis because of an effect
called the Nuclear Overhaus Effect (NOE). Hence these peaks in the C13 spectrum

cannot be integrated for calculation of DS or extent of positional substitution.

Figure 6.11 shows the C13 spectrum of starch propionate of DS 3.0. This spectrum
had the same peaks as that of the unmodified starch except for two distinct peaks
occurring at a ppm of 170-175 and two more at approximately 8ppm and 26ppm.
The former are the carbonyl peaks, and the latter are the methyl/methylene signals
from the acetyl/propyl group substituted in the starch. The leftmost peak had a
height and integrated area almost twice that of the second one. This is because the
C2 and C3 carbons have very similar molecular surrounding, as a result of which
the carbonyl peaks from the acetyl groups substituted at these carbons have
approximately the same chemical shift. The C6 carbon has a slightly different
environment and so has a carbonyl peak slightly shifted from the other two
carbonyl peaks. Figure 6.12 shows the C13 spectrum of starch acetate of DS 2.0.
The difference between the acetate and propionate showed up in an additional
methylene signal and a shift in the carbonyl peak further down for the propionate.

The C6-OH group is theoretically the most reactive and thus the more easily
substitutable one than the other two OH groups. So, when the starch is partially
acetylated, this difference should show up on the C13 NMR spectrum. Figures
6.13 and 6.14 show the carbonyl peaks of starch propionate with a DS of 1.0 and

1.7 respectively. In the case of the cellulose acetates, the cellulose is first

86

——-172 907

 

 

..'.. “I'LL... in.“ "MIMI 1w". ,.,.,.. ' 'W .' ..,“‘

178 177 176 175 174 173 172 171 170 169 00m

Figure 6.13 - C13 NMR of starch propionate DS1.0 (Carbonyl peaks)

““173 190

D
D
a,
n
h
ve

 

 

 

I I'I'IIIIVI rvYfi—

I I ' I l r
I
178 177 176 175 174 173 172 171 170 ppm

Figure 6.14 - C13 NMR of starch propionate DSl.7 (Carbonyl peaks)

87

 

 

 

 

 

Lula...“-

mrllIIIIIII[IIIIIIIII]IIIIlIIIIIIIIIIIIII'IIIIWIIIIII'IIIIIIIII'ITIIIIIIIIIIIIIIIII'IIIIIIIIIIIIIIIIIII'IIIIII

 

 

200 180 160 140 120 100 80 60 40 20 O ppn
S
r: rx
2 as
g r
8
3
o;
2

 

 

 

 

 

 

 

 

I I l I I I I I I I IfT I I VI I
172 171 170 169 168 167 166 165 1

Figure 6.15 - C13 NMR of cellulose acetate DS 2.5

88
acetylated completely and then back hydrolyzed to give the desired DS. So, in this
case, most of the carbonyls should occur at the C2, C3 positions. Figure 6.15
shows the spectrum of cellulose acetate of DS 2.5. A comparison between the
cellulose acetate DS 2.5 and starch acetate DS 2.0 (Figure 6.12) shows a distinct
difference in the position of the carbonyl hyperfinely split peaks on the spectrum.

Detailed interpretation of the spectrum was not done in this work.

6.4 CORRELATION OF BIODEGRADABILITY WITH POSITION
AND DEGREE OF SUBSTITUTION

It has been mentioned in literature that biodegradability is affected by the position
of substitution. According to Bhattacharjee et a1. [Bhattacharjee (1971)], work
done on Carboxy-methyl cellulose showed that chain scission takes place most
readily between two adjacent unsubstituted residues but, nevertheless, can occur at
a residue having a 6- or a 2- substituted algycone. For e.g. a derivative with a D8
of 2.0, and having 2- and 3- substituted would show a retarded rate of degradation
as compared to a derivative with a D8 of 2.0, but with 6- and 2—(or 3-) substituted.
Although, it was intended to explore this area further in this work, it could not be
done. This is an important area forfurther research, since it relates back directly to
the method of synthesis of the derivatives, i.e., whether they are stoichiometrically
derivatised to the desired DS or completely derivatised and then back hydrolyzed
to the desired DS.

The degree of substitution affects the rate of biodegradation of the starch and
cellulose esters to a major extent. It was found that the higher the degree of
substitution, the lower is it’s rate of biodegradation. This area has been studied in
detail in Chapter 7.

'l Chapter 7

 

STARCH ESTERS

 

7.1 BACKGROUND

Starch is a natural polymer which is readily biodegraded in both aerobic and
anaerobic environments to form smaller end products. Unfortunately it cannot be
used as it is for the production of plastic materials owing to it’s high moisture
sensitivity and poor thermal processibility. Modification of natural polymers
through the acetylation or propionation of the available hydroxyl groups permits
the formation of appropriate film-forming plastic copolymers. However, the
technical difficulties associated with the development of a biodegradable product
are numerous. Environmentally non persistent materials are required to have both
stability during the useful lifetime of the product and performance characteristics
that the public has grown to expect while being rapidly degraded in biologically
active environments, such as waste water treatment or composting facilities. These

product requirements often seem mutually exclusive [Buchanan et a1. (1992)].

The biodegradation of modified starches and celluloses of DS>1 is a hotly debated
field. Very little experimental work has been done in this area. Biodegradation of
these materials has been studied at very low DS levels within the solubility range
of the material, but not at high DS levels. The relationship between the degree of
substitution of starch esters and it’s biodegradability has been studied a little

89

90
previously, but under different environmental conditions. Rivard et al. did some
work on various modified natural polymers and their fate in an anaerobic
environment. In their work, cellulose, starch, and xylan were substituted with
acetate to various degrees, and the effect of this modification on the anaerobic
biodegradation assessed through a biochemical methane potential (BMP) protocol.
Significant reduction in anaerobic biodegradability resulted with all polymers at
substitution levels of between 1.2-1.7. In all the three cases, biodegradability was
reduced to about 10% after a substitution level of 1.7 was reached. Similar work
was done by Bhattacharjee et al. [Bhattacharjee (1971)] on cellulose ethers. He
reported that cellulose ethers with a DS greater than 1.0 generally do not
biodegrade while those with a D8 of less than 1.0 will degrade due to attack of

microorganisms at the unsubstituted residues of the polymer.

In this work, starch propionates of different DS were synthesized and tested for
their aerobic biodegradabilities in the presence of municipal sewage sludge as per

the procedure described in Chapter 3. The results are reported in this chapter.
7.2 SYNTHESIS OF THE MODIFIED STARCHES

The chemically modified starches were synthesized at Michigan Biotechnology
Institute by the Biomaterials group. Starch propionates of different DS were
synthesized by the following procedure [Methods in Carbohydrate Chemistry, V].

The glass reactor was assembled as shown in Figure 7.1. 25g of the Hylon VII
starch sample was weighed out and mixed with 100 ml of DMSO in a 250 ml

Erlenmeyer flask on a stir plate to make a fine paste.

91

I Port 3

N2 purge « Port 2
Port 1 V o \

 

 

Glass Reactor

 

 

Agitator

 

 

 

Figure 7.1 - Glass Reactor Assembly for synthesis
of modified starches

92
Care was taken while using the hygroscopic chemicals to keep moisture absorption
to a minimum while being transferred to the reaction vessel. The slurry was added
to the vessel through port 1 using a short stemmed funnel. The agitator was turned
at medium speed to attain good mixing without spattering the liquid. The
Erlenmeyer flask was rinsed out with another 100 ml of DMSO which was added
to the reaction vessel through the same port. Using the silicone oil bath, the vessel
was heated to 110°C, and the temperature inside the vessel was kept below 90°C.
At the end of the heating period, the solution was a clear translucent liquid, yellow
liquid. The vessel was cooled to room temperature using a water bath. A solution
of DMAP (Dimethyl amino pyridine) and 50 ml of DMSO was prepared. Amounts
of catalyst, sodium bicarbonate and the anhydride were dictated by the moles of
starch and the degree of substitution. The catalyst was 4 mol% of the anhydride.
the sodium bicarbonate was added using a powder funnel through port 1. The
moles of sodium bicarbonate added was equal to the anhydride. DMAP solution
was added through the same port using the same funnel as the bicarbonate. The
solution was allowed to stir until there were no clumps of sodium bicarbonate left.
A solution of propionic/ acetic anhydride was prepared in 100 ml of DMSO and
added using a dropper funnel at the rate of 1-2 drops per second. After addition of
the anhydride, the reaction was allowed to proceed for 15 min. The product was
precipitated in water, blended and filtered. The product was washed five times or
until the pH of the solution was the same as the original wash water. The product

was filtered and then allowed to dry in a vacuum oven.

Starch propionates of different degrees of substitution were prepared and tested for

their biodegradabilities under a given set of experimental conditions.

93
7.3 BIODEGRADATION OF STARCH PROPIONATES WITH
VARYING DS

7.3.1 Materials tested
The CHN data of these materials were determined using the Perkin Elmer 2400
CHN Elemental analyzer. The CHN data on the materials tested are given below.

Sarnplel - Starch propionate, DS = 1.0 (sample AG-2-14-92), %C = 47.13, %H =
6.57, %N=0.14.

Sample2 - Starch propionate, DS = 1.5 (sample AG-10-23-92, 12L scale-up ), %C
= 50.15, %H = 6.45, % N = 0.17.

Sample 3 - Starch propionate, DS = 1.7 (sample ADL 9-30-92, lst 10 gallon scale-
up), %C = 51.22, %H = 6.62, %N = 0.15.

Sample 4 - Starch Propionate, DS = 2.7 ( sample AG-10-21-92), %C = 53.93, %H
= 6.50, %N = 0.14

Sample 5 - Cellulose (Microcrystalline Avicel) used as a positive standard. Aldrich
Chemical Company, 20 micron particulate solid, %C = 39.98, %H = 18.37, %N =
0.076.

The DS of all the starch propionates were determined through proton NMR. The
NMR spectra of these materials are presented in Figures 7.2 -7.5

7.3.2 Inoculum and test solution
The test materials were of known weight based on their carbon contents. The C/N
ratio maintained in each flask was 36:1. This was maintained by adjusting the

amounts of stock solutions used.

94

 

 

 

 

 

J

IIIIIIIIIIIIIIIIIIIIIIIIIYWIIIIIIIIIIITHIIIIIITIIII'IIIITI

6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
L ' ‘1' ____1____1

 

 

44.56
19 C"

Figure 7.2 - Proton NMR of Starch propionate DS = 1.0

 

 

 

 

 

 

 

 

 

 

IIIIIIIIIIIIIITIIIIII'IIIIIIIIIIIIIIIIIITIIIII‘IIiIIIIIfiI—IIIIT

6.5 6.0. 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 pp!

'——_—-J

 

"4.16
‘-“.;4

Figure 7.3 - Proton NMR of Starch propionate DS = 1.5

95

__.__—_._.

/

/-

/
/

T I I I 1 I I I I if I I T I T T I I T I T I I I I
5 4 3 2 1
. __,_r —__,___.
44.79

]

 

 

 

 

 

31.47

(31.46

Figure 7.4 - Proton NMR of Starch Propionate DS = 1.7

 

 

 

 

 

 

 

 

‘
p

 

U
s:

 

Figure 7.5 - Proton NMR of Starch Propionate DS = 2.76

96
The amounts of test materials used were as follows :-
Sample 1 = 3.39 g, C content = 1.6 g
Sample 2 = 3.19 g, C content = 1.6 g
Sample 3 = 3.12 g, C content = 1.6 g
Sample 4 = 2.97 g, C content = 1.6 g
Cellulose = 4.00 g, C content = 1.6 g
The stock solutions used in each flask were as mentioned in chapter 3, except that
5 ml of ammonium sulfate was added. 30 ml of the sludge inoculum was used.
Water was added in the flasks and the level brought up to about 1500 ml. The N in
the sludge was negligible and not taken into account. The total N in the solution
was calculated as 0.0451 g.
The C/N ratio in the solution thus worked out to be 35.5.

7.3.3 Results and Discussion

The experiment was carried out in the manner described earlier. Figures 7.6 and
7.7 show the percentage carbon converted to CO; with time for all the samples
tested. From the two figures, it can be seen that the standard, cellulose reached a
C02 evolution of 77% in a period of 40 days. It was found that for all the
materials the rate of C02 evolution reached a maximum in the first 5-10 days. The
test was run for a period of 50 days. The results are shown in Table 7.1. The
modified starches took off to a slow start, and the extent of biodegradation in the
given time frame decreased with increasing degree of substitution. The
experimental correlation of the DS and extent of biodegradation is shown in Figure
7.8. Starch propionate of DS 1.0 reached a biodegradation extent of 35%, whereas
the DS 3.0 reached a biodegradation extent of only 7%. Figure 7.9 shows the

experimental correlation of DS and percentage biodegradation relative to

97

 

 

 

 

1.0'
N ..
8 0.8 t I . Cellulose
3 . .. 0 DS 1.2
t 1
g I ‘ DS 1.6
2 0.61 I 0 DS 1.4
§ ' I DS3.0
U
"5 0.44 '
.5. ’ . °
‘5 a o
g I
1‘" 0.2J II . ..
I . / _
/ ’ I '
0.0 A . . . .
0 20 40

Time (Days)

Figure 7.6 - Percentage of initial polymeric C converted to CO, with
time for starch propionates of varying DS. Cellulose is included as
the positive control. Each curve represents an average of duplicate
results. No blank correction has been applied to the above data.

98

 

 

 

0.51
N
O
U
c 0.4‘
; u
0 I
t I I I D812
2 0-3‘ . DSl.4
3 ' . DSl.6
U 02. I ~' 0 . 1383.0
0 .
DD
5 ‘ 0
n I
8 01~ - ..
23 / 0
a. t

./
0.0 ‘/ . . - T '
0 20 4O
Time(Days)

Figure 7.7 - Percentage of initial C converted to C02 with time for starch
propionates of varying DS (Cellulose excluded). This figure is a replicate
of Figure 7.6 with the cellulose curve excluded from the graph.

99

 

starch

 

 

Percentage C converted to C02 relative to

o
qp
I»

p
.L
db
4-

 

0 ' 0.5 1 1.5 2 2.5 3
Degree of substitution

 

 

 

Figure 7.8 - Relation between biodegradation and degree of
substitution. Percentage of polymeric carbon converted to
CO, at the end of 48 days was plotted for starch propionates
with different degrees of substitution relative to percentage C
conversion to C02 for unmodifiedstarch (DS=0) which was
scaled to 100%.

100
unmodified starch (unmodified starch was run in the next set of experiments,

where the standard cellulose had the same rates as this set).

Table 7.1 - Summary of biodegradation results

 

 

 

 

 

 

 

 

 

 

Materials %C converted to CO;

Cellulose 78%

SP -DS 1.0 32.5%

SP-DS 1.5 15%

SP—DS 1.7 12%

SP-DS 3.0 7%
5333213?) 70%

 

SP - Starch propionate

7.3.3 Conclusions

These results clearly show that in an aerobic environment, the biodegradability of
starch propionates decreases with increasing DS. The decrease in the extent of
biodegradation with increasing chemical modification in other environments have
been given different explanations. All of them however relate to the accessibility
of the starch or cellulose backbone (the 1-4 linkage) by the degrading enzymes.
This inhibition may be either a result of the increased hydrophobic nature of the
modified polymers, which reduces wetting and thus enzyme/microbe surface
contact, or the stearic hindrance presented by the modifying groups on the
polymer, which reduce enzymatic activity. Accessibility of the polymer backbone
to the enzymes has also been related to. morphology and the porosity in literature.
These factors have been investigated experimentally in Chapter 8.

101
7.3.4 Limitations of the above data
The experiment I‘conducted on the starch propionates was one of the first
biodegradability experiments carried out in this work. Hence, it had some
drawbacks which are listed below.
a) A large shaker was used for all the test flasks instead of individual stir plates for
each of these flasks. The large shaker did not provide adequate mixing between the
materials and the inoculum. Glass beads were added to the solutions to improve
the mixing. This was achieved but still not to a satisfactory level.
b) Some problems were encountered in the air flow system which affected the air
flow rates. This was because of a lot of back pressure inside the flasks. The flasks
were sealed with tygon tapes to prevent them from popping. There was a positive
pressure at the inlet and the outlet of the trapping solutions was exposed to air.
This created some problems in the flow lines, as a result of which the air flow

rates WCI'C uneven.

Chapter 8

 

CELLULOSE AND CELLULOSE
ACETATES

 

8.1 BACKGROUND

The technical difficulties associated with designing biodegradable polymers are
extremely complex. Biodegradable polymers must be both cost effective and have
acceptable performance characteristics, typical of common synthetic polymers
and, at the same time, they must be non-persistent in the environment [Buchanan
et al.(1993)]. Viewed from current technology, these requirements are often
mutually exclusive. Based on this view point, cellulose esters offer a very
attractive potential in terms of material properties and environmental persistence,
since it is derived from a natural polymer, cellulose, which is readily biodegraded

in the environment.

Cellulose is readily biodegraded in both aerobic and anaerobic environments to
yield smaller end products which are assimilated into the nature’s carbon cycle.
However, cellulose is not readily processable, since it’s melting temperature lies
above it’s decomposition temperature [Buchanan et al (1992)]. This is a common
problem with unmodified naturally occurring polymers. The biodegradability of
cellulose esters has been a widely debated topic. Derivatisation of cellulose

improves it’s thermal stability with lower melting temperatures, but increases it’s

102

103
resistance to biodegradation. In order to understand the mechanism of

biodegradation of cellulose acetate, it is essential to understand it’s structure.
8.2 STRUCTURE OF CELLULOSE ACETATE

The structure of cellulose and cellulose acetate is shown in Figure 8.1. The
cellulose acetate polymer is basically a random copolymer of mono, di, triacetate
and unsubstituted monomer units on the polymer backbone. In the case of
cellulose acetate DS=2.4, the di and tri substituted units predominate in the
polymer. It has been found that substitution at the 2- and 3— positions affect the
chain scission at the glycosidic bond. Cellulose acetate will be referred to as CA in

this chapter.
8.3 Cx - C1 MECHANISM OF CELLULOSE BIODEGRADATION

The enzymatic hydrolysis of cellulose is complex owing to three major factors
[Focher et a] (1991)] -

o Heterogeneity of the system

0 Dual nature of the insoluble substrate (crystalline and amorphous)

0 component multiplicity of the enzymatic system
The mechanism of enzymatic hydrolysis of cellulose has been explained in
principle, in terms of the sequential action of three different enzyme complexes
[Shirnada and Takahashi (1991)] which are -

o C,, Endo-cellulase, catalyses random hydrolysis of B-l,4 glycosidic

linkages of inner amorphous regions of a cellulose chain in a random

manner

104
Structure of Cellulose

“'— 1,4-B linkage

CHZOH K
o
W\
CHZOH

 
    

 

 

— n

Structure of cellulose triacetate monomer

C —— O

C/ 5 \C
4
__l \C|5_(|32/ CL.

0 Ac OAc
Structure of cellulose acetate of DS 2.4
(acetyl content = 40.2)

fH20ACO\ fI'IIOI'I fflzofl
<f°\ (“C [6“ ”5} °
OAc OAC OAc OAc '0“ I0" iAc/ OH

Figure 8.1 - Structure of Cellulose acetate-random copolymer of
monoacetate, diacetate, triacetate and unsubstituted units

 

 

 

Insoluble cellulose

‘ (DP>6)

 

 

 

 

 

% $5:- % g g Q Soluble cellulose
C1 (DP<6)
@ Q % fl
B—gluoosidase
\— Cellobiose mainly
(DP=2)

l

assesses $3,

 

Figure 8.2 - Cx - C1 mechanism of enzymatic hydrolysis

106
0 C1, Exo-cellulase, catalyses the endwise hydrolysis of B-l,4 glycosidic
linkages, in principle splitting off cellobiose product from the non
reducing end of the crystalline cellulose chain.
0 B-glucosidase catalyses the hydrolysis of the B-glycosidic linkage of
cellobiose units.
The enzymatic hydrolysis of cellulose takes place by the Cx - C1 mechanism as

shown in Figure 8.2, which involves the synergistic action of the three hydrolases.
8.4 CA BIODEGRADATION LITERATURE

Some work has been done on the enzymatic degradation of cellulose ethers, and it
has been seen that the degree of substitution is the most important factor in the
biological degradation of cellulose ethers. Similar work has also been done on
some cellulose esters yielding similar results. It was found that when the D8 of the
cellulose derivative increased to above zero, the biodegradation rates decreased
rapidly, to the extent that there was almost no biodegradation reported for D8 of
1.0. Carboxy methyl cellulose was studied extensively for it’s biodegradation by
Bhattacharjee et al. (1971), and it was found that since the unsubstituted residues
are the main focal point of attack, there needs to be at least two unsubstituted
residues for hydrolysis (chain scission) to occur. In related studies on
hydroxyethyl cellulose [Wirick et al. (1968)], it was found that chain scission
takes place readily between two unsubstituted residues, but nevertheless can occur
at a residue having a 6— or 2- substituted aglycone. Rivard et al. conducted studies
on the anaerobic bioconversion of cellulose acetates to methane, and reported that

the biodegradability of the acetates decreases with increasing DS and shows

107

 

Cellulose Acetate Biodegradation

 

o Biodegradation '5 dependant on degree of substitution

- Low DS cellulose derivatives readily biodegradable
- High DS (> 2.0) cellulose derivatives biodegradable ??

/ \

/ NEGATIVE

 

 

( POSITIVE

   
     
    
 

 

 

   

- Buchanan et al. (1993) - Rivard et al. (1992)
0 Gross et al. (1993) . Barenberg et al. (1991)
- Glasser et al. (1993)
0 Gilmore et al. (1991)
Proposed mechanism Pr0posed mechanism
Deacetylation

Cell- Ac ‘7’? Glucose-Ac

cam ——> Cellulose

1

C02 ‘— Glucose C02

Inacceseibility of substrate
(b-l,4 linkage) to enzymes

 

 

..........

.....
_.;.;. ......
. -.-.’Z- .........

Figure 8.3 - Summary of the conflicting reports on CA biodegradation

108
negligible biodegradation after a D8 of 1.5-1.7. Similar results were reported by
Weirner et al. Some studies were also conducted on blends of high DS cellulose
acetates and propionates with PHB/V [Gilmore et a1 (1993)]. It was shown that
both environmental and enzymatic assays on the blend films showed a strong
inhibitory effect of cellulose acetate butyrate or cellulose acetate propionate on
PHB/V degradation: the weight loss of the films never exceeded 10% when the
cellulose ester content was 50% or higher. In contrast to the above results,
Buchanan et al.., conducted studies on cellulose acetates and propionates and
showed that DS of as high as 2.5 in the case of acetates and 1.7 in the case of
propionates underwent rapid biodegradation in an aerobic enriched environment.
The conflicting reports and proposed mechanisms on CA biodegradability are

summarized in Figure 8.3.

This chapter deals with the biodegradation of cellulose acetate of DS 2.5 obtained

from two different sources - Eastman Kodak and Hoechst Celanese.

8.5 CA BIODEGRADATION :- EXPERIMENTAL

8.5.1 Materials tested

Two samples from different sources were tested for their biodegradability. The
molecular weights of the samples were obtained from Eastman Chemical Co. The
D8 of these materials were determined by proton NMR on a VXR 500. The carbon
contents of the material were obtained through a Perkin Elmer 2400 CHN
Elemental analyzer. The two samples of cellulose acetate were CA398-30 and
JLF68.

109
CA398-30 :- This sample was obtained from Eastman Chemical Co., and had a
molecular weight of around 30,000, and a D8 of 2.5 as determined by proton
NMR (Figure 8.4). It was sieved to obtain a fraction between mesh size 120(.007
inches and mesh size l40(.003 inches). It had a bulk density of 80 lb/cu.ft.. It had
a %C=48.59, %N=0.023, %H=5.89.
JLF68 :- This sample was obtained from Hoechst Celanese. It was a plastics grade
product with a molecular weight of around 30,000, and a DS of 2.49 (acetyl value
of 55.5%) as determined by proton NMR (Figure 8.5). It was sieved to obtain a
fraction between mesh size 120 (0.007 inches) and mesh size 140 (0.003 inches). it
had a packed bulk density of 26.0 lb/cu.ft. It had a %C=48.04, %N=0.007,
%H=5.88.
A summary of the material properties is given in Table 8.1

Table 8.1 - Material Properties of the CA samples

 

 

 

 

 

 

CA 398-30 ”“8
SOURCE Eastman Kodak Hoechst Celanese
(Mn)* 58,000 62,000
(Mw)* 161,000 188,000
Polydispersity 2.76 3.04
C content 48.59% _ 48.04%
DS 2.5 2.5

 

 

 

 

110

 

 

______’———~

/

 

 

 

 

 

 

. I
l
:3 L

r T r I v 1 , . I Y ' ‘ r T
3 2 . 1

 

 

Figure 8.4 - Proton NMR of CA-398-30

 

 

 

 

 

 

 

 

d h

I I T I I I '
6 . 5 4 3 2

L‘

l .
‘l

H

 

Figure 8.5 - Proton NMR of JLF-68

111
8.5.2 Inoculum and Test solution
The test materials were of known weight based on their carbon contents.
CA398-30 = 3.09 g, carbon content 2 1.5 g
JLF68 = 3.12 g, carbon content = 1.5 g
Cellulose = 3.76 g, carbon content = 1.5 g
The stock solution used in each flask were as mentioned in chapter 3 except that 5
ml of ammonium sulfate was added. 30 ml of the sludge inoculum was added and
water was added to the flask upto a level of 1500 ml. The C/N ratio maintained

was approximately 33/1.

8.5.3 Results

The biodegradation results of this experiment are shown in Figures 8.6 and 8.7.
Figure 8.8 shows the same results but with no blank correction. This experiment
was run for 40 days, during which time, the % C conversion to C02 was 0.48 for
CA398-30 and 61.17 for JLF68. The standard cellulose reached a C02 evolution
level of at the end of 40 days. As can be seen there was a considerable difference
between the two CAs, which was surprising since they were essentially the same
material with the same properties (Table 8.1). When the samples were examined
under the scanning electron microscope, it was found that the materials had a very
different morphology. Bulk Density data from the supplier’s specifications also
showed some difference in values. The differences between the two samples are
summarized in Table 8.2.

8.5.3.1 Scanning electron Microscopy - Figures 8.9 and 8.10 show the SEM
micrographs of the two CA samples taken by a JEOL 35CF Scanning Electron
Microscope. The powders were first mounted on a platinum stub and carbon paint

was applied at the edges of the stub to improve conductivity. The samples were

112

 

70¢.

a,

o
A
v

 
 
 
  
   
   
  

33

E

 

+ Cellulose
+ 11.1368
+CA398-30

i

 

 

 

Percentage C converted to C02
8

 

 

 

 

Figure 8.6 - Percentage of initial polymericC converted to
C02 with time. Percentage C conversion to CO, for two CA
products of DS 2.5 was measured with time. Cellulose was
included as a positive control. Each curve represents the
average of duplicate runs.

 

113

 

 

3.00 q - + cell“.

 

 

 

 

Rate of C converted to C02 in mg/hr

 

 

 

 

Tirm in days

 

 

Figure 8.7 - Rate of initial polymericC conversion to C02
with time. Rate of carbon conversion to C02 for two CA
products of DS 2.5 was measured with time.

114

 

80¢.

 

 

 

 

Percentage C converted to C02

 

 

 

Tine indays

 

 

 

Figure 8.8 - Percentage of CA carbon converted to CO, with
time (no blank correction). This figure is a replicate of Figure
8.6 except that no blank correction has been applied.

0081 10.0U CE094

Ivy ‘4- A
V"; -31: 7
[fl .

_ -

2.0UP 10.00kU 2.92kx

 

X—Z3433—093—4 10-13—1933 7:22

Figure 8.9 - SEM micrographs of CA-398-30 at 3000K

116

18 . BU CE094

2.92kx

2.08P 10.00kU

:ZS

 

7
3
9
9
1
_
3
1
_
U
1
4..
3
9
n_u

X—23433

Figure 8.10 - SEM micrographs of J LF-68 at 3000X

117
then coated with gold on a sputter coater for 3 mins (21A thickness approx). An
accelerating voltage of 10kV, working distance 15 or 35 and condenser lens setting

of 400 or 600 was used depending on the magnifications used.
8.6 EFFECT OF MORPHOLOGY - LITERATURE

It has been well established that for any substrate to be available for microbial
attack, a direct contact needs to be established between the acting enzyme and the
active site region. Cellulose has been studied to some extent as far as morphology
effects are concerned, but cellulose derivatives have been studied very little. This
area has been explored in terms fiber size, pore size etc., of the cellulosic substrate

[Tanaka (1983), Wong (1988)].

Table 8.2 - Differences between the two cellulose acetates

 

CA-398-30 JLF-68

 

BULK DENSITY 801b/cu.ft 26 lb/cu ft.

 

 

MORPHOLOGY Closed Structure smooth Open honeycomb structure
surface

 

 

 

They have shown that the course of enzymatic solubilization of cellulosic
materials is governed by the surfaceharea of pores accessible to the enzymes.

Moreover, only small cellulase components can diffuse slowly into small pores

 

118

 

       
 

CA-398r30
closed structure

 

 

      
 

.....................................
. . . . . . . . . . . . . . . . -.-.- . . . . . . ....r. . . . .... .. ....
..............................................................................

Precipitation
in water ’

 

‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘
.............................
.............

JLF-68

open, honeycomb ¢
structure

 

    
     

 

    

Water washing
Vaccum filtration

V '-'.-'.-I-2-‘.-2-Z-I‘C-C-i-f-I-I'I-Z'2'2-1'1'1-3'1-2-2'2'1'2'1-1-2':{-Z-Z'I'I-Z-I-I-Z-C-I-Z{-l-l'Z-I-Z'I-Z-I-I- ........
...................................................................................................................

 

Size reduction
Seiving
'3313:5313?i237:3:3:33121:13::-'EIEtiiiiiiééiiiiiéiiifiiifiiéii2i:‘2I::i:i:5:i:j:é:é2:3?€6§§§¥§

  

 

r

   
 
   
      

Closed cell structure
(confirmed by SEM)
DS = 2.5 (NMR) ’2’”;

i

Biodegradation
Assay

w. - -' .3;,;.::;:_~:-:;:_-:-:-:;:;:-_:;:;:;:;:;:;:;:§:§:::§:§i§:§:§:f:::;:;:;:;:§:§:§:§:§i§f§§

 

 

C02 evolution

    
  
    

 

 
 

Extent of biodegradation
- % C conversion to C02

Figure 8.11 - Preparation of samples with defined morphology

119
inaccessible to the entire complex of enzymes, and once they enter in they are
trapped there and thus prevent further molecular movement and synergistic action.
The biodegradability of cellulose derivatives has also been explored in terms of the
charge on the substrate. It has also been shown that positively charged cellulosic
substrates attract enzymes more than the negatively charged ones [Boyer (1983)],
which in turn affects the rate of hydrolysis.

8.7 CA WITH DEFINED MORPHOLOGY -EXPERIMENTALS

8.7.1 Preparation of CA samples with defined morphology

CA 398-30 and JLF68 were separately dissolved in acetone, and precipitated in
water. The precipitates were thoroughly washed with water, vacuum filtered and
dried. They were then ground to obtain a fine powder and sieved to obtain a size
fraction between 120 and 140 mesh size. Figure 8.11 summarizes the procedure
followed for the sample preparations. The D8 of these two samples were obtained
through proton NMR. They both had a DS of around 2.5 (Figure 8.12 and 8.13).
SEM pictures were also taken and revealed a similar closed morphology for both
the samples (Figure 8.14 and 8.15).

8.7.2 Inoculum and Test Solution

CA398-30 (ppt) = 1.6 g, carbon content = 0.8 g

JLF68 (ppt) = 1.6 g, carbon content = 0.8 g

Cellulose = 1.5 g, carbon content = 0.8 g

The stock solution used in each flask were as mentioned in chapter 3 except that 3
ml of ammonium sulfate were added. Water was added to the flask upto a level of
1500 ml. The C/N ratio maintained was approximately 33/ 1.

120

 

 

 

 

 

 

r..—
WTIfiI— l—rfirw I I 'Ijr I ' I I ‘ I—‘1_'-'—r| TI |
55 50 45 4.0 3.5 30 25 20 1.5 1(

 

 

Figure 8.12 - Proton NMR of precipitated CA-398-30

 

 

 

 

 

at.

"—1—‘—|' ‘—'l ” l—"T' 1 1"I—I" 'I '—I" ' _I ""I -1" I —| f‘I—‘1' 'I "'1 I‘-'I I T _'| -

6 . 5 4 3 2 1

#

 

 

 

Figure 8.13 - Proton NMR of precipitated JLF-68

121

 

10u

 

Figure 8.14 - SEM micrograph of precipitated CA398-30 at 3000X

 

122

 

 

 

 

Figure 8.15 - SEM micrograph of precipitated JLF-68 at 3000X

123

 

 

70--

 

 

 

 

Percentage C converted to C02

 

Tine in days

 

 

 

 

 

 

 

 

 

 

15 20

 

Rate of C converted to C02 with
time

Tim in days

 

Figure 8.16 - CA- carbon conversion to C02 with time
for the precipitated CAs. Carbon conversion to CO2 was
measured for the precipitated CA products with similar
morphology. Each curve represents an average of
duplicate runs.

 

124

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

909
8
80”
U
3 70..
'U
3 600
g +Cellulose
>
g 50‘ +CA3983ppt
u 40- +prt
U
3., 30-
S
5 20d A
2
u 10-
an
0 ‘ t i :
0 5 10 15 20 25 30 35
'Ilrmindays
2..
+Cellulose
N 1.80
O +CA398.3ppt
U 1.6--
3 +JLF68ppt
g 1.40
h 12«
o
z 1‘
8 0.81
U 06
$5 .
3 0.4a
00
a 021
A
° 4 : fi‘
0 5 10 15 20 25 30 35
'lineindays

 

 

 

Figure 8.17 - CA carbon conversion to CO, for the
precipitated CAs (no blank correction). This figure is a
replicate of Figure 8.16 except that no blank correction has
been applied here.

125
8.7.3 Biodegradation Results
Figures 8.16 and 8.17 show the biodegradation results of the above experiment.
Table 8.3 summarizes the biodegradation results of both the experiments. Both the
materials reached approximately the same C02 evolution level within the time
frame of the experiment which was 35 days. This confirmed the fact, that the
morphology of the material played an important role in the biodegradabilities of

the two materials tested here.

Table 8.3 - Biodegradation results - Percentage C converted to CO; with time

 

 

 

 

Unprecipitated (39 days) Precipitated (33days)
Cellulose 60.14 (69.1) % 62.94 (82.07) %
JLF68 61.17 ( 70.17) % 0.28 (19.07) %
CA398-30 0.48 (9.48) % 0.07 (18.86) %

 

 

 

 

 

The bracketed values are the ones with no blank correction

8.8 DISCUSSION

The difference in the biodegradation rates of the two different morphology
samples of cellulose acetate may be related to the accessibility of the polymer
backbone active sites to the degrading enzymes. It is seen that the cellulose and the
JLF68 biodegradation curves are very similar. This may be an indication that since
JLF68 is very porous, the active sites on the cellulose acetate backbone are easily
accessible to the enzymes, and hence undergoes biodegradation with the same
kinetics as the cellulose itself. This theory is supported in part by a work done

earlier by Fujii et al. on cellulose derivatives in which he refers to the molecular

126
weight of the polymer as a limiting factor instead of the porosity. He claims that
below a certain molecular weight the reaction kinetics of the cellulose derivatives
are the same as that of pure cellulose. On the other hand, CA398-30 has a closed
structure which does not allow the enzymes to access the active sites on the
polymer backbone. This limits the rate of hydrolysis of the cellulose backbone and

also the extent of hydrolysis.
8.9 PROPOSED MECHANISM FOR CA BIODEGRADATION

Two mechanisms have been proposed for the biodegradation of Cellulose acetate
of high DS. They are:
a) Chain scission of the polymer backbone followed by assimilation of the

monomer glucose acetate (N arayan et al).

cellulose 0| igom er
———>
Acetate Acetate

l

Glucose
Acetate

1

(:02

Figure 8.18 - Proposed mechanism for aerobic enzymatic degradation of CA

b) Deacetylation followed by chain scission of the resultant polysaccharide

backbone and assimilation of the generated glucose units (Buchanan et al).

127
The results obtained from the morphology studies suggest that the accessibilty of
the polymer backbone may be a key factor in the biodegradability of the high DS
cellulose acetate. Some initial studies were conducted on the samples during the
biodegradation experiments. It was found that after a 35% C conversion to C02
was reached for the CA, the D8 of the sample (extracted, freeze-dried and studied
through NMR), was slightly more than the original value, i.e., DS = 2.7. This
suggested that after the unsubstituted units along the CA backbone were attacked,
further C02 generation was obtained by chain scission of the CA backbone. Based
on these findings, the first mechanism was proposed by Narayan et al, which is
schematically represented in Figure 8.18. This is only a hypothesis and needs to be

investigated further for confumation.

The key points in this hypothesis are as follows -
- Cellulose acetate is “intrinsically” biodegradable - i.e., it is capable of
being utilized by microorganisms with no gratuitous or co-metabolism.
0 Deacetylation may not be essential for biodegradation.
- Accessibility and transport of enzyme to the active site is the rate
lirnting step for biodegradation of cellulose derivatives.
0 Degree of substitution is not the rate limiting factor except as it modifies
the enzyme transport mechanism.
However, to claim that CA is biodegradable in composting or waste disposal
systems,
0 CA must be engineered to biodegrade in the operating time frame of the
disposal system (composting, sewage/waste water treatment facilities,

soil, etc.) to an appreciable extent, greater than 50%.

128
0 Trace the disposition of the residual carbon - cell mass, humic
substances, oligomers and determine the rate of biodegradation and time

taken for complete biodegradation/assimilation.

In order to create a kinetic model for the the proposed biodegradation mechanism
of cellulose acetate, cellulose degradation kinetics were studied theoretically as a
first attempt. A kinetic model was developed for the aerobic biodegradation of

cellulose in the experimental test flask.

8.10 PROPOSED KINETIC MODEL FOR THE AEROBIC
BIODEGRADATION OF CELLULOSE

8.10.1 ASSUMPTIONS
A set of assumptions were imposed in deriving a comprehensive mechanistic
model for the aerobic enzymatic hydrolysis of cellulose. These assumptions were

based on Fan and Lee (1983) , as well as P.L. Beltrarne’s (1984) assumptions.

1) The hydrolysis of cellulose occurs in two steps: the insoluble cellulose is
hydrolysed first to soluble cellulose by the synergistic action of endo-glucanase
and cellobiohydrolase, and the cellobiose is further hydrolysed to glucose by the
action of b—glucosidase. The first two enzymes have been lumped together as a

single enzyme complex, B, and the third enzyme referred to as KB-

2) The two fractions of cellulose, the crystalline and the amorphous are not
distinguishable.

129
3) The overall biodegradation rate is not affected by diffusion or mass transfer,

i.e., the product concentrations are governed by the reaction kinetics.

4) The rate of glucose production is equal to the rate of glucose cosumption to give
C02. This assumption was based on negligible glucose concentrations of samples
taken out from the experimental flasks periodically during the period of
biodegradation. This has been explained further.

5) The hydrolysis of cellulose to cellobiose occurs at the cellulose surface by the
adsorbed enzyme. This reaction is non-competitively inhibited by cellobiose. No
inhibition by glucose takes place owing to negligible concentration of glucose in
solution. The decomposition of cellobiose to glucose takes place in the aqueous

phase, and following the previous assumption, is not inhibited by glucose.

8.10.2 COMPETITIVE AND NON-COMPETITIVE INHIBITION

The difference between competitive and non-competitive inhibition is shown in

'3Q ‘

Non-competitive Conpetitive

Figure 8.19 - Competitive and Non-competitive inhibition

130
Figure.8.19. Competitive inhibition is a situation where there is an inhibitor
molecule binding to the enzyme active site, thus blocking it, so that the substrate
molecule cannot bind on to the same active site. Non-competitive inhibition on the
other hand, binds away from the active site, thus giving free access to the active
site for the substrate. In both the cases, there is a reduction in the affinity of the

enzyme for the substrate, but no reduction in reaction rates.

8.10.3 MECHANISM OF DEGRADATION
The assumptions in this model gives rise to two major steps in this overall

reaction.

8.10.3.1 Decomposition of cellulose to cellobiose
The decomposition of cellulose to cellobiose is a heterogeneous reaction, which
includes the adsorption of the enzyme, and the formation of the enzyme-substrate

complex. The following reactions are taking place in this step.

 

 

 

 

k
E+S —-'-‘ ES
K1
(1)
_1<2..
ES+C k ESC
'2 (2)
k2
E+C EC
192
(3)
_1<3_.
ES E+C

(4)

131
where,E = lumped enzyme complex
S = cellulose substrate
ES = enzyme-substrate complex
C = cellobiose
EC = enzyme-cellobiose complex
ESC = enzyme-substrate-cellobiose complex in non-competitive inhibition
by cellobiose
k1 and k1 = forward and reverse rate constants for equation (1)
k; and k2 = forward and reverse rate constants for equation (2) and (3)
k3 = rate constant for equation (4).
Equations (1) and (2) represent the decomposition to give cellobiose, whereas (3)

and (4) represent the inhibition reactions by cellobiose

8.10.3.2 Decomposition of cellobiose to glucose
In this step the decomposition of cellobiose to glucose takes place in the aqueous
phase, and includes the hydrolysis of the cellobiose to glucose with no inhibition

from glucose. The following reactions take place in this step

 

$0..
Es": 1., E06
(5)
c k4 G
E —> +
B E" (6)

where,G = glucose formed
E8 = enzyme B-glucosidase

EB C = enzyme-cellobiose complex.

132
k4 = the rate constant for equation (6).

kg and k_p L—l forward and reverse rate constants for equation (5)

8.10.4 DESCRIPTION OF THE MODEL

The following mathematical assumptions are made for the model.

1) Equations ( 1) and (2) and (3)are in equilibrium and the equilibrium constants
are given as,

K1=£ K2=_lf_2.

k k
'1 ‘ (7)

Instead of equilibrium, we can also assume that equations, (1) and (4) follow a

pseudo-steady state condition, i.e.,

$155-
dt ’0

k: (E) (S) - k1 (ES) - k3 (ES) = 0

However, k3 is relatively very small, i.e., the reaction (4) is a very slow one.

Hence, the approximation that equation (1) is in equilibrium has been used.

2) The complexes, (EC), and (ESC) are dead end-complexes, i.e., they do not
break down to give the product.

133

3) The reaction (5) is in equilibrium, and the equilibrium constant is given by,

KB = _kfl
k—B
(8)

Instead of equilibrium, we can also assume that equations (5) and (6) follow a
steady state condition, as in the case of (1) and (4). But since, k3 is very small, the

equilibrium assumptions is applied.

3) The process setup is a batch reactor with the schematic shown in Figure 8.20.

 

 

 

 

Air
————>
Enzyme +
Substrate Air + (:02
—>
Balsam

Figure 8.20 - Process Schematic for the batch reactor
The assumption made here is that the rate of production of glucose by the
enzymatic hydrolysis equals the rate of consumption of glucose by aerobic

conversion to C02, i.e.,

Rate of C conversion to CO; = rate of C production as glucose.

134
Since, the glucose produced is rapidly being removed from the solution as C02,
the inhibition by glucose in both steps of the mechanism have been disregarded.
Thus,

d CCO2 dC glucose

 

 

8.10.5 DEVELOPMENT OF THE MODEL
8.10.5.1 Reactions with enzyme E
The reaction mechanism mentioned earlier has been schematically depicted in

Figure 8.2].

 

£——'£C

+5

 

 

ES‘I—i‘fisc

l

E+C

 

Figure 8.21- Reaction mechanisms for E and EB

a) From equation (1),

k1(E) (S) =k_l(ES)

K £1.=_@§)_
1( k.1 (E)(S)

From equation (2),

k 2 (ES) (C) =- 1220380

_}_<_2_ _ (ESC)

K = _
2 122 (ES) (C)

From equation (3),

k 2 (E) (C) =--- k_2(EC)

k
K2 = _2 (EC)

122 ._._. (E) (C)

A total enzyme balance in the batch reactor for E yields

Et =E+(EC)+(ES)+(ESC)

where, E. = total enzyme E in the batch reactor.

From equation(7),

_ EL
(E) " K1(S)

(9)

(10)

(11)

(12)

(13)

136

From equation ( 8)

(ESC) = K2 (ES) (C) (14)

From equation (7) and (9),

K2 (C) (ES)
K 1 (S)

 

(EC) =
(15)

Substituting equations (11), (12), (13) in equation (10), we obtain

 

_ (gs) K2(C)(ES)
lat - 19(5) + K16) + (ES) +K2(ES) (C)

Er

 

_2_
k1(S) '1' K1 (S) + 1 + K2(C)

 

(16)

Thus, we get, from equation (4), the concentration profile of the intermediate

 

product C with time as,
k E
3 t
dC
— = k =
dt 3 (BS) 1 K (C)

 

__2_
k1(S) + K1 (S) + 1 + K2(C)

(17)

137
8.10.5.2 Reactions with enzyme EB

The reaction mechanism mentioned earlier has been depicted in Figure 8.22.

+C

 

 

Efic—"EB+ C

EB

(18)
Figure 8.22 - Reaction scheme for EB
From Eq. (5),
R [3055) (C) = k_B(Eg3 C)

KB = 1239
(EB) (C) _ (19)

Also, from a total enzyme balance E3; in the batch reactor, we get,

EBt = (EB) + (BBC) (20)

So, from (17) and (18), we get,

 

_ 1
Eflt — (BBC)(KB(C)+ 1)

Thus, we get from equation (6), a concentration profile for the final product

glucose as,

138

dG k4EBt
v: — — k4(EBC) : 1

t ( +
KB (C)

 

 

1 )
(21)

8.10.5.3 Mass balance (grams of carbon) in reactor
A carbon balance can also be done in the batch reactor to account for the

concentrations of substrate(cellulose), cellobiose and glucose as,

SO=S+C+G (22)

where, S0 = concentration of substrate at time, t=0

8.10.5.4 Summary
We thus have three variables S, C and G, and the three equations,

k3 Et

 

dC
—— = k (ES) =
dt 3 1 K (C)

k1(S) + —2—Kl (S) + 1 + K2(C)

 

dG k4EBt
V: — = k4(1‘313C)=(1

dt +
Kp (C)

 

 

1)

So=S+C+G

These three equations can be solved simultaneously to give the concentration

profiles with time.

Chapter 9

 

CONCLUSIONS AND
RECOMMENDATIONS

 

The current focus of the materials industry, specially packaging and disposables,
on the redesigning of materials keeping in mind their ultimate environmental fate,
provided the thrust for this work. Studies were conducted on the aerobic
biodegradation of polymeric materials in the presence of municipal sewage sludge

on a laboratory scale.

A viable method was evolved based on ASTM-D5209 to quantitatively measure
the aerobic biodegradation of polymers in a liquid inoculum derived from
municipal sewage sludge. It utilized the polymeric carbon as a single carbon
source producing carbon dioxide which was periodically measured. This method
was used successfully to quantify the extent of biodegradation of polymers based
on percentage of original carbon conversion to C02. The results from all the
experiments were reproducible within a maximum of :l: 10% of the presented
results. The cellulose standard showed consistent carbon dioxide evolution levels
for all the experiments within the experimental time frames applied (5 60% within
30-35 days with blank correction) thus confirming the viability and reproducibility
of the inoculum used. The rate of C02 evolution was also found in all cases, to

reach a maximum within the first 7 - 10 days from the start of the experiment, and

139

140
then slowly lag off to a steady rate after 25 - 30 days. This can be attributed to the

growth cycle of the microorganisms present.

A theoretical analysis was done of the biodegradation process and the effect of
certain process parameters on the biodegradation measurement around the
experimental system. The effects of speed of agitation and inoculum
acclimatization were studied experimentally. It was found that the extent of
biodegradation increased with increasing speed of agitation. This was because of
increased contact, hence increased surface area exposure of the materials to
microorganisms with increasing agitation speed. Acclimatization of the inoculum
was found to appreciably decrease the standard deviations between the replicates
(< 3%), but did not increase the extents of biodegradation. Acclimatization for a
longer period of time and serial transfers could result in a more uniform and
concentrated inoculum with respect to the favorable microorganisms. This is an
important area for further investigation. Another parameter that can be investigated
further is temperature since this would raise up questions about the type of

organisms actively involved in the biodegradation processes.

Several commercial as well as other polymers of interest were tested for their
biodegradabilities and the results reported. Cellulose, Starch, 9-11 acid and
glycerol triacetate showed appreciable biodegradation extents of >60%. PCL and
the PCL/starch blend had relatively slower but steady C02 production rates and
reached biodegradation extents of s 40% within the experimental time frame.
PVOH and EVOH showed very little biodegradation contrary to reported
literature. This could be due to the insolubility and the high crystallinity of the two

materials. Aqua Coat was found to show low levels of biodegradation, indicating

141
that when coated on paper, it will retard and maybe limit the biodegradation of
otherwise naturally biodegradable paperboard. This material was studied because
of the large use of coated paperboard as food cartons and other packaging material

in the food industry.

Considerable attention was paid to the cellulose and starch esters, owing to their
potential as biodegradable plastics. Both these chemically modified esters are
economically attractive owing to their natural abundance and possess good
mechanical and plastic properties comparable to most of the commercially
available plastics. Starch propionates of different DS were characterized and tested
for their biodegradabilities. It was found that the biodegradation decreased
considerably with increasing DS owing to increased hydrophobicity and decreased
accessibility of the active sites by the degrading enzymes. The correlation of
biodegradability to positional substitution and method of synthesis is an interesting

topic for further investigation.

Morphology was found to affect the biodegradation of cellulose acetate DS = 2.4
to a great extent. A CA sample with a porous, honeycomb structure was found to
have a much higher biodegradation rate as compared to a CA sample with a closed
smooth morphology. Re-precipitation of both the samples and reproduction of the
closed smooth morphology resulted in almost negligible biodegradation. It was
therefore concluded that the accessibility of the polymer backbone (i.e., the
glycosidic bonds) is the governing factor in the biodegradation of the starch and
cellulose esters. Based on these conclusions a mechanism was proposed for the
biodegradation of CA which involved polymer chain cleavage, followed by

monomer/oligomer assimilation by the microorganisms. As a first attempt, a

142
kinetic model was developed for the aerobic biodegradability of cellulose under

the given experimental conditions.

In order to track down the mechanism of CA biodegradation experimentally,
periodic measurement of chemical changes need to be done during the
biodegradation assay. This can be done by periodic extraction and freeze drying of
the CA samples from the experimental test flask followed by characterization
using NMR for determining DS and GPC for determining molecular weight. An
enzyme assay could also provide some interesting insights into the biodegradation

mechanism.

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