A Handbook of Applied Biopolymer Technology: Synthesis, Degradation and Applications
A Handbook of Applied Biopolymer Technology: Synthesis, Degradation and Applications
A Handbook of Applied Biopolymer Technology: Synthesis, Degradation and Applications
Series Editors:
James H Clark, Department of Chemistry, University of York, York, UK
George A Kraus, Department of Chemistry, Iowa State University, Iowa, USA
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-FP001
A Handbook of Applied
Biopolymer Technology
Synthesis, Degradation and Applications
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-FP001
Edited by
Sanjay K. Sharma
Jaipur Engineering College & Research Centre, Jaipur, Rajasthan, India
Ackmez Mudhoo
Department of Chemical and Environmental Engineering,
University of Mauritius, Reduit, Mauritius
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A catalogue record for this book is available from the British Library
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Preface
interruptions and damage. With this in focus, this book provides an up-to-
date, coherently written and objectively presented set of book chapters from
eminent international researchers who are actively involved in academic and
technological research in the synthesis, degradation, testing and applications of
biodegradable polymers and biopolymers. Hence, the overall pool of latest
ideas and recent research and technological progress achieved in the synthesis,
degradation, testing and applications of biodegradable polymers/biopolymers
together with a high level of thinking have been presented in a comprehensive
perspective to make progress in the emerging field of biodegradable polymer
science and engineering (or bio-based polymers). The element of environ-
mental sustainability as linked to biopolymer technology also constitutes the
essence and novelty of this very relevant book in today’s era of environmental
depredation.
This book consists of book chapters written and contributed by international
experts from academia who are world leaders in research and technology
regarding sustainability and biopolymer and biodegradable polymer synthesis,
characterization, testing and use. The book highlights the following areas: Green
polymers; Biopolymers and bionanocomposites; Biodegradable and injectable
polymers; Biodegradable polyesters: Synthesis and physical properties; Discovery
and characterization of biopolymers; Degradable bioelastomers, Lactic acid-
based biodegradable polymers; Biodegradation of biodegradable polymers;
Biodegradation of polymers in the composting environment; and Recent research
and application development in biodegradable polymers. The book is aimed at
technical, research-oriented and marketing people in industry, universities and
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vi Preface
institutions. The book will also be of value to the worldwide public interested in
sustainability issues and biopolymer development and as well as others interested
in the practical means that are being used to reduce the environmental impacts of
chemical processes and products, to further eco-efficiency, and to advance the
utilization of renewable resources in bio-based production and the supplier chain.
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-FP005
The main outcomes of reading this book should be that the reader will have a
comprehensive and consolidated overview of the immense potential and ongoing
research in bio-based and biodegradable polymer science, engineering and
technology, which is earnestly attempting to make the world of tomorrow
greener. Hence, this handbook is a reasonably comprehensive and applied treatise
of the topic and provides up-to-date information to a very wide audience on the
applied research areas of biopolymers.
Sanjay K. Sharma
Ackmez Mudhoo
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Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-FP007
Ackmez Mudhoo
For you Neelam.
Sanjay K. Sharma
This book is for Kunal and Kritika, my twin angels.
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Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-FP009
Contents
1.1 Background 1
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2.1 Introduction 22
2.2 Triglycerides of Fatty Acids and their Derivatives 24
2.2.1 Monomers from Triglycerides 24
2.2.2 Polymers Synthesized from Triglycerides 29
2.3 Essential Oils, Natural Phenolic Compounds and
their Derivatives 34
2.3.1 Terpenoids 35
2.3.2 Phenylpropanoids 40
2.3.3 Lignin Digests or Extracts and
Liquefied Wood 46
2.3.4 Other and Natural Phenolic Compounds 48
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x Contents
2.4 Carbohydrates and their Derivatives 51
2.4.1 Polymers from Popular Carbohydrates 51
2.4.2 Furan Derivatives 54
2.5 Monomers Obtained by Fermentation 55
2.6 Conclusions and Outlook 60
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-FP009
References 61
3.1 Introduction 79
3.2 History of Polyhydroxyalkanoates 80
3.3 Chemical Organization of PHAs 80
3.4 Occurrence and Biosynthesis of PHAs 81
3.5 Cheap Substrates for Cost-effective PHA Production 86
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Contents xi
4.3.3 Starch-based Green Composites 117
4.3.4 Soy Protein-based Green Composites 117
4.3.5 PLA-based Green Composites 118
4.4 Applications 121
4.5 Conclusion 122
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-FP009
References 123
xii Contents
Contents xiii
References 305
xiv Contents
11.3 Composting Process Essentials 339
11.3.1 Composting Chemistry 339
11.3.2 Physical Parameters in Composting 344
11.3.3 Composting Systems 345
11.3.4 Vermicomposting 345
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-FP009
Contents xv
Chapter 13 Impacts of Biodegradable Polymers: Towards Biomedical
Applications 388
Y. Omidi and S. Davaran
xvi Contents
14.4 Injectable Ceramics 429
14.5 Injectable Cell Vehicles 431
14.5.1 Naturally Derived Hydrogels 432
14.5.2 Synthetic-based Hydrogels 433
14.6 Injectable Drug Delivery Systems 434
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-FP009
Prof. (Dr.) Sanjay K. Sharma is a very well known author and editor of many
books, research journals and hundreds of articles from last twenty years.
Dr. Sharma did his Post Graduation (1995) and Ph.D. (1999) from the Uni-
versity of Rajasthan, Jaipur. His field of work was synthetic organopho-
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xvii
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(Rajasthan) India.
Mr Ackmez Mudhoo, presently Lecturer in the Department of Chemical and
Environmental Engineering at the University of Mauritius, obtained his
Bachelors degree (B.Eng. (Hons.)) in chemical and environmental engineering
from the same university in 2004. He then read a master of philosophy
(M.Phil.) degree by research in the same department from 2005 to 2007. His
research interests encompass the bioremediation of solid wastes and waste-
waters by composting, anaerobic digestion, phytoremediation and biosorption.
Ackmez has 48 international journal publications (original research papers,
critical reviews and book chapters), 5 conference papers to his credit, and an
additional 7 research and review papers in the pipeline in his early career.
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1.1 Background
Hidden in an old Art Nouveau townhouse in Essen, Germany, is the world’s
largest collection of plastic artifacts. This is the private Kölsch Collection,
created by the architects Ulrich and Ursula Kölsch, which houses tens of
thousands of plastic items, all numbered and placed on shelves from floor to
ceiling in every room of the ground floor flat. With every single item perfectly
and painstakingly catalogued by Mr and Mrs Kölsch, the collection is the most
complete archive of the developments in the plastics industry over a 150-year
span. We not only see the development of plastics, but also how this unique
material helped in the development and change of design and aesthetics. The
Kölsch’s collection shows us how, in the first 80 years of the plastics industry,
products were made exclusively of biopolymers, most from renewable resources
such as cellulose, casein, shellac and ebonite. Here and there, we stumble upon
items made of plastic materials that most of us have never even heard of, such
as Bois Durci and kopal resin. However, these materials, long dropped from
our collective memories, helped shape what is today the plastics industry.
For example, Bois Durci or Hardened Wood, an early plastic that dates back
to the 1850s, is a mixture of sawdust (usually of a hardwood such as ebony or
1
2 Chapter 1
rosewood), carbon black, metal particles and blood or egg. The sawdust was
mixed with vegetable oils, mineral or metallic fillers, and the blood with a
gelatinous substance diluted in water. The dry and wet components were mixed
and compressed into finished parts in a steel mold held in a steam-heated screw
press. Figure 1.1 shows a French mirror made of this early plastic material by
compression molding using a screw press.
Continuing the walk through the Kölsch’s art townhouse, we find colorful
kopal artifacts brought to us in the Art Deco period between 1921 and 1931 by
the EBENA company in Belgium. Also molded in a heated screw press, these
items, most of which are beautiful decorative Art Deco bowls, containers, boxes,
lamps, radios (Figure 1.2), and clocks, are made of a mixture of wood fiber,
pigments and a fossilized tree resin from Congo called kopal. Kopal is found in
the ground in the jungles of Africa. It can be considered to be the inland
counterpart of amber, which is found in the North and Baltic Seas. The EBENA
factory in Wijnegem, Belgium, existed between 1921 and 1931, and was the only
manufacturing facility to ever produce kopal products. Since EBENA closed in
1931, the expensive fossilized resins such as kopal and amber have been for-
gotten as molding materials, except for perhaps a few jewellery applications.
In their collection, it is not difficult to stumble upon items made of casein, a
milk protein obtained via acidification or enzymatic action. The resulting curds
can be dried, molded, and treated with a hardening agent to yield commercial
plastic. Casein had its heyday as a commercial plastic in the early 1900s; the
same time period when many other items that are found in the Kölsch’s
collection were produced.
Figure 1.1 Bois durci mirror from France, circa 1880 (Kölsch Collection).
History of Sustainable Bio-based Polymers 3
Figure 1.2 Jewellery box molded from kopal resin by EBENA (Kölsch Collection).
The recent revival of interest in these materials makes the story of their
discovery and development even more relevant today. The great success of
petrochemical-based polymers from the Second World War through the
present is testament to the versatility, economy, and durability of such synthetic
materials. However, the indestructibility that for decades made petrochemical-
based synthetics so desirable has increasingly become a liability. Over 25 mil-
lion tons of plastic entered the municipal solid waste stream in the USA in 2001.
This non-biodegradable plastic waste accounted for over 11% of the municipal
solid waste in the USA, up from 1% in 1960.1 Incinerating plastics can cause
toxic air pollution; plastic litter is unsightly. Thus there are health, environ-
mental, and aesthetic problems with continued use of non-biodegradable
petrochemical-based polymers. The expanded use of renewable, biodegradable
biopolymers would alleviate the problems associated with disposal of non-
biodegradable polymers. In addition, the increasing monetary and political
costs of American and European dependence on foreign sources of oil make
sustainable, domestically grown resources a desirable alternative.
Renewed interest in biopolymers since the mid-1990s has shown itself in
new research into the processing and properties of renewable, biodegradable
materials like casein, soy protein, polylactic acid (PLA), polyhydroxyalk-
anoates (PHA), and other novel materials. This work has demonstrated the
opportunity for renewable, biodegradable biopolymers to replace their syn-
thetic counterparts in a variety of applications. But the interest, needs, and
materials that are re-emerging are a continuation of a story that began centuries
earlier; the opening chapter of this ongoing story is told here.
This chapter presents various biopolymers within a historical perspective in
relation to today’s needs. We will cover silk, casein, and soy, as well as other
4 Chapter 1
materials that have recently been found useful in recent applications such as
chitin, collagen, PHA, and PLA.
OH
H H H
O O CH3 O CH3
N N N
N N N
O O O n
H H H
Figure 1.3 Repeating chemical structure of silk fibroin, composed of the amino acid
sequence: glycine-serine-glycine-alanine-glycine-alanine.4
History of Sustainable Bio-based Polymers 5
bleeding) were still popular two centuries later, as reflected in one of Shake-
speare’s comedies, A Midsummer Night’s Dream, where he wrote: ‘‘I shall desire
you of more acquaintance, good master cobweb. If I cut my finger I shall make
bold with you . . .’’.7
Around the end of the eighteenth century, it was established that bleeding
vessels were better treated by ligatures (tying up the ends of the vessels) than by
cautery (burning the ends of the vessels). By this time, waxed thread had been
replaced by silk as the material of choice. Philip Syng Physick (1768–1837) was
an American who became the first professor of surgery at the University of
Pennsylvania. Following the teachings of his mentor, the famous John Hunter,
a Scot who became the founder of experimental surgery and surgical pathology,
Physick used adhesive leather strips to close a wound. He then noticed that
these dissolved in contact with fluids from the wound. He thought this char-
acteristic would be of great advantage in the use of ligatures. This idea was
historic, since no one had previously thought of a suture that would be
absorbed after performing its function.
In 1867, Joseph Lister, among his great contributions in antisepsis, wrote an
article ‘‘Observations of ligature of arteries on the antiseptic system’’. He
believed that a silk ligature could be left in the body if bacteria lying within the
interstices of the threads could be killed. At that time, ligatures were left long
and protruding through the wound to then be pulled out along with the
necrosed or dead tissue at the end of the vessel, increasing the risk of a sec-
ondary hemorrhage. In his experiments, he started using antiseptic silk ligatures
soaked in an aqueous solution of carbolic acid, where he found the ligature was
not absorbed after ten months of implantation on the external iliac artery of a
50-year-old woman. These results lead him to explore, in 1868, the use of ox
peritoneum and catgut in a carbolic acid solution, seeking an antiseptic
absorbable ligature.
In 1881, arguing that carbolized catgut (referring to Lister’s mixture of olive
oil and carbolic acid) was not an effective antiseptic, Kocher of Berne started a
campaign against catgut and in favor of silk. In his rules of surgery, Halsted,
who introduced thin rubber gloves in 1890, recommends: ‘‘. . . gentle handling
of tissues, meticulous haemostasis, and interrupted silk sutures’’.
By 1900, however, the catgut industry was firmly established in Germany,
using the intestines of sheep, important in their sausage industry.5 Nevertheless,
both catgut and silk were important base materials for the production of
sutures for the following 100 years.8
By then, the silk industry was large and of great economic importance, as silk
was being used for a large variety of consumer goods, from clothing, weavings
and stockings for women. However, most of the silk, the raw material for this
growing industry, came from Japan and China, a relatively unstable part of the
world in the first part of the twentieth century. This prompted the growing
Western industries to concentrate on finding replacements for silk, as had been
done with natural rubber, through chemistry. The downfall of silk as an
industrial material began in 1927, when the DuPont Company hired the
chemist Wallace Hume Carothers to run their ‘‘pure research laboratories’’.
6 Chapter 1
The exit road for silk was paved by 1938, a year after Carothers’s death, when
nylon was introduced to the world, primarily as a replacement for silk in hose
and stockings and as toothbrush bristles. It is certain that the invention of
nylon gravely affected the Japanese trade balance, and in consequence, the
overall position of the Japanese industry in world markets at the threshold of
the Second World War. The influence of this miracle fiber, that could be
produced at the fraction of the cost as its natural counterpart, is indisputable.
Allied use of nylon in parachutes during the invasion of Normandy may have
played a decisive role in the war’s military outcome. The most obvious influence
may come from its impact on consumer consumption.
As time progressed, the news for silk turned even more dire; in the 1960s, the
use of virgin silk was found to produce an adverse biological response in
sutured patients. This was later attributed, in the late 1970s and early 1980s, to
sericin in the inner fibroin fibers of the silkworm silk, which was found to cause
a type I allergic reaction. Virgin silk was then processed to extract sericin from
its fibroin fibers, followed by a coating of wax or silicone to improve material
properties and reduce fraying, and received the name of black braided silk (e.g.
Perma-Handt). Due to the biocompatibility issues, however, between the
1960s and the 1980s, silk decreased in its popularity as a suturing material.9–11
In addition, these events ran in parallel with the development of synthetic
biocompatible polymers, based on polyglycolic acid (PGA) (Dexont,
Maxont) and polylactic acid (PLA) (Vicrylt). These two, together with catgut
(Catgutt), are classified as biodegradable suture materials, according to the
definition of an absorbable suture material by the US Pharmacopeia: one that
loses most of its tensile strength within 60 days after being placed below the
skin surface.8,12 Silk-based sutures, along with other kinds based on braided
polyester (Ethibondt, Mersilenet, Tevdekt), nylon (Ethilont) and poly-
propylene (Prolenet, Surgilenet), are classified as non-absorbable suturing
materials.12 Some studies on silk, however, have showed its susceptibility to
proteolytic degradation and the loss of the majority of its tensile strength in vivo
after 1 year of implantation.13–16 The braided structure of silk-based sutures
increases the risk of infection, but these sutures have great handling and tying
capabilities, and therefore it is still used today around eyelids and lips, where
incidence of infection is low.8
Today, silk-based biomaterials are reviving, accompanied with advances in
molecular and genetic manipulations. Its great mechanical properties and
degradation characteristics have opened doors in the fabrication of tissue
engineering scaffolds, which need ample time to interact with the host tissue
before degrading. Some studies have found silk scaffolds comparable to those
based on collagen for culturing bone and ligament tissue, as well as fibroblasts
and bone marrow stromal cells.12,17–19 The capability of processing silk
fibroin into foams, meshes, fibers, and films make silk a promising material
for several biomedical applications.12,20,21 Advancements in genetic manip-
ulation and protein tailoring have included spider silk in this array of
opportunities, offering even superior mechanical properties when compared
to B. mori silk.20–22
History of Sustainable Bio-based Polymers 7
1.3 Cellulose: The Quintessential Bio-based Plastic
If we step back to the nineteenth century, another natural polymer, cellulose, in
addition to rubber, impacted everyday life. The invention of cellulose plastics,
also known as Celluloid, Parkesine, Xylonite, or Ivoride, has been attributed to
three people: the Swiss professor Christian Schönbein, the English inventor
Alexander Parkes, and the American entrepreneur John Wesley Hyatt.
Christian Friedrich Schönbein, a chemistry professor at the University of
Basel, loved to perform chemistry experiments in the kitchen of his home, much
to his wife’s dismay. Early one morning in the spring of 1845, Schönbein spilled
a mixture of nitric and sulfuric acids, part of that day’s experiment, on the
kitchen counter. He quickly took one of his wife’s cotton aprons and wiped the
mess up, then rinsing it with water before the acid would damage the cloth. As
he hung the apron to dry over the hot stove, it exploded in a loud bang and
flame in front of his very eyes. After he recovered from the shock, Schönbein’s
curiosity led him to impregnate wads of cotton with the acid mixture. Every
time, he was able to ignite the mass, leading to an enormous, uncontrollable
explosion. He called his invention guncotton. He had invented cellulose nitrate.
Guncotton was three times as powerful as gunpowder and did not leave a black
cloud after the explosion. Schönbein sold his patent to the Austrian Empire’s
army, but found no buyers in Prussia, Russia, or France. Finally, he sold his
patent to John Taylor, his English agent, who immediately began production of
guncotton in England. The production ended when his factory exploded, killing
20 workers. Although there were no buyers, several laboratories did spring up
across Europe to investigate guncotton; often blowing up faster than they were
being built. In addition to its military applications, Schönbein envisioned other
uses for the nitrated cotton mass. He added a solvent or plasticizer made of
ether and alcohol and found a way to nitrate the cellulose fibers into a less
explosive material which he called kollodium, glue in Greek. He reported to his
friend Michael Faraday that this mass ‘‘is capable of being shaped into all sorts
of things and forms . . .’’. In the spring of 1846, after accidentally cutting
himself on the hand, he covered the wound with a thin elastic translucent film
made of kollodium. He sold his idea to the English, who for years supplied the
world with the first adhesive bandages. In England, there was one person that
took particular interest in the Swiss professor’s inventions. His name was
Alexander Parkes.
Alexander Parkes started playing around with cellulose nitrate in 1847, and
spent the next 15 years in the laboratory perfecting the formulas and processes
to manufacture cellulose nitrate. His final process took the nitrated cotton and
added vegetable oils and organic solvents producing a ‘‘plastic mass’’ that was
easily molded into any shape or form after it was softened under heat. He called
his plastic mass Parkesine. The new applications for this versatile material, such
as combs, knife handles, and decorations, made their debut at the 1862 World
Exposition in London. In 1866, Parkes launched the Parkesine Company Ltd.
Due to the low quality of its products, Parkesine was not a success and the
company was liquidated in 1868. The poor mixing of the additives and solvents
8 Chapter 1
caused Parkesine products to significantly warp only a few weeks after man-
ufacture. In 1869, Parkes sold his patents to Daniel Spill, his chief engineer,
who founded the Xylonite Company and renamed the compound Xylonite.
Parkes continued working on his material until his death in 1890 at the age
of 77. Alexander Parkes, the inventor and engineer, can be credited with
improving on Schönbein’s invention, paving the road for the future of the
plastics industry. He is also credited with fathering a total of 20 children. A very
busy man, to say the least.
At the same time as the plastics industry seemed to be going under in Eng-
land, in the United States John Wesley Hyatt was launching an enterprise that
finally made cellulose nitrate a success, under the name of celluloid. As the
story goes, it all began when in 1865 the billiard ball manufacturer Phelan &
Collendar placed an ad that promised $10 000 to the person who would find a
replacement for ivory in the manufacture of billiard balls. Elephants were being
slaughtered at a rate of 70 000 per year, which would have led to the extinction
of this great animal, exorbitant prices for the ‘white gold’ from Africa, and
reduced profits for the billiard ball industry. The $10 000 prize attracted the 28-
year-old Hyatt’s attention. After returning home from his job as a printer, he
worked on this project until eventually he stumbled upon nitrocellulose in 1869.
After finding a better way to mix all the components as well as allowing the
solvents to completely evaporate from the mass before solidification, he was
soon manufacturing high-quality billiard balls. Instead of cashing in on the
$10 000 prize, John Hyatt founded the Albany Billiard Ball Company with his
brother Isaiah, becoming a direct competitor to Phelan and Collendar. For the
next 30 years, until Bakelite replaced celluloid on the billiard table, many guns
were pulled in the Wild West when the volatile balls sometimes exploded upon
collision.
Another immediate application of celluloid was dentures, which up until then
were made of hard rubber. In view of losing a rather profitable business to
plastics, the rubber industry started a propaganda campaign against cellulose in
all major US newspapers. They falsely claimed that celluloid dentures could
easily explode in one’s mouth when coming in contact with hot food. This not
only cheated people of a much prettier smile, but also started a rivalry between
the two industries, which has caused them to remain as completely separate
entities to this day. In fact, despite the materials and processing similarities
between plastics and rubber, the plastics industry and the rubber industry have
completely separate societies and technical journals. A plastics engineer is likely
to be found in meetings organized by the Society of Plastics Engineers (SPE) or
the Society of the Plastics Industry (SPI), while a member of the rubber
industry will attend meetings organized by their own society, the Rubber
Division of the American Chemical Society.
With a new and versatile material, Hyatt and his co-workers needed equip-
ment to mass-produce plastic products. Based on experience from metal
injection molding, the Hyatt brothers built and patented the first injection
molding machine in 1872, to mold cellulose materials,23 as well as the first blow
molding machine, to manufacture hollow products. In the summer of 1869,
History of Sustainable Bio-based Polymers 9
Hyatt and Spill, respectively, filed for patents dealing with the manufacture of
nitrocellulose materials. This started a lengthy and costly litigation that even-
tually ruled in Hyatt’s favor in 1876. Spill died soon after, at the age of 55, of
complications from diabetes. John Wesley Hyatt lived another 44 productive
years in which he invented the injection molding and the blow molding
machines with which he processed celluloid products. The combination of blow
molding and celluloid resulted in many toys and household products such as
jewellery boxes (Figure 1.4). Hyatt can certainly be credited for being the first
person to successfully mold a plastic mass into a useful, high-quality final
product. However, above all, we should credit him for saving the elephant on
the road to a $10 000 prize he never claimed.
With the mass production of rubber, gutta-percha, cellulose, and shellac
articles during the height of the industrial revolution, the polymer-processing
industry after 1870 saw the invention and development of internal kneading
and mixing machines for the processing and preparation of raw materials.24
One of the most notable inventions was the Banbury mixer, developed by
Fernley Banbury in 1916. This mixer, with some modifications, is still in use
today for rubber compounding.
Figure 1.5 Spitteler’s cat ‘‘inventing’’ casein plastics (courtesy of Luz M. Daza).
History of Sustainable Bio-based Polymers 11
27,28
applying pressure and shear, as in a heated extruder. Water was added
to casein resins as a plasticizer prior to molding. Water enhanced proces-
sability, but resulted in greater shrinkage and warpage upon drying.26 The
same gains in processability could be achieved by using a 10% borax
solution in place of the water.28 This reduced shrinkage, but the finished
material still easily absorbed large amounts of moisture and was therefore
subject to significant warpage. This shortfall was addressed by ‘‘hardening’’
the casein plates and rods in a 40% formaldehyde solution, rendering them
water insoluble.28 Even so, the water absorption of casein was approxi-
mately 20–30%.26
The development of casein in the USA proceeded more slowly than in
Europe. It was not until 1919 that the first casein plastic that was up to Eur-
opean quality standards was made in the USA.26 Even then, the industry in the
USA did not take off for several reasons: climatic differences meant the Eur-
opean processes could not be copied in a straightforward manner in the USA,
the long and costly process did not yield easily to faster processing in automatic
machines, and the scrap could not be reworked, so 50% waste was not
uncommon. All this made competition with established materials more
difficult.26
In 1929, Christensen added aluminum stearate, a water-soluble aluminum
salt, to the resin and found that a non-hardened plastic rigid enough to be
worked in automatic machines was produced. This not only allowed the casein
plastic to be worked in machines, but aluminum casein scraps were easily
re-workable, greatly reducing waste.26 In 1926, 55% of the world’s buttons
were made of casein.26
Casein production in the 1930s steadily increased, from 10 000 tons produced
worldwide in 1930 to 60 000 in 1932 and 70 000 in 1936.26 However, buttons,
belt buckles, jewellery, and ornaments (Figure 1.6) remained the main products
in which casein could be used, pending a method of reducing the absorption of
water from humid air.26 Additionally, casein plastic still required hardening in
formaldehyde, making its manufacture a long and costly process. The hard-
ening and subsequent drying could take anywhere from two weeks to a year,
depending on the thickness of the part.29
An event that led to further improvements occurred in 1938 in Utica, New
York, when a tannery was ordered to shut down because it was polluting a local
stream. William S. Murray, a prominent Utica politician and chemist, obtained
a 30-day stay and found a method of solidifying the waste runoff, thereby
protecting the stream. He applied the same methods to the skimmed milk being
discarded by the nearby powdered milk plant. By changing the natural milk
sugar to an aldehyde in the presence of casein, he found a method of casein
plastic production that avoided formaldehyde hardening.30 He secured a
patent, but the Second World War prevented any further development of his
promising new process.31 The continued use of phenol formaldehyde and
widespread adoption of synthetic plastics during and after the Second World
War drastically curtailed the use of casein, which is produced at a minimal level
today.25
12 Chapter 1
Figure 1.7 Henry Ford sits behind the wheel of a car whose body panels are made
from a soy protein-based plastic (courtesy of the Henry Ford Museum).
Figure 1.8 Electron micrograph of collagen fibers (reproduced with permission from
ref. 54).
The use of natural polymers for the culture of cells is advantageous due to the
better capability of cells to attach and interact with particular amino acid
sequences. However, there is great variability from batch to batch,59 and syn-
thetic polymers have found their way into the tissue engineering scaffold world.
The properties of synthetic polymer scaffolds are more controllable and
reproducible, but a great portion of research has been necessary to account for
optimal cell interaction.59 Several studies have combined natural polymers or
critical amino acid sequences with synthetic polymers to find an ideal cell–
scaffold relationship. Although synthetic materials are still on the rise for use in
tissue engineering scaffolds, much attention is steered towards the use of nat-
ural polymers. Growing knowledge about genetic and molecular manipulation
allows researchers to tailor some of the properties of these proteins, as well as
blend them with other synthetic materials in search of the ideal tissue engi-
neering scaffold.
1.8 Conclusions
From the Second World War through the 1980s there was little research done on
bio-based plastics. However, research in these materials is experiencing a
resurgence. Environmental concerns, on the part of both developers and con-
sumers, associated with petrochemical plastics have led to an increased interest
in environmentally and biofriendly alternatives, despite their high cost in com-
parison with established synthetic materials. Additionally, great advances in
biomedical research, genetics, and molecular biology have directed much
attention to biodegradable and biocompatible natural polymers. These advances
have also allowed the development and growth of green industry with bacteria
as producers of raw materials that substitute for petroleum-based resources.
Walking down a quiet street in Essen and through the door to the Kölsch’s
collection is more than just paying a visit to the past. It is also passing through
the doorway to a place where time remains frozen, to help us step into the future
where renewable, as well as biocompatible, materials will help us free ourselves
from fossil fuel dependence and will usher us into a sustainable industrial age,
and will bring us opportunities and solutions in the biomedical field.
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CHAPTER 2
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2.1 Introduction
Synthetic polymer materials made from petroleum chemicals have become
major materials over the past 50 years, because of their durability, plasticity,
low cost, mechanical flexibility, abundant variety, lightness, and so on.1 They
play very important roles in many industries to produce not only daily com-
modities but also technological devices and biomedical devices. Behind their
convenience, however, the abundant use of synthetic polymer materials has
brought gradual spreading of durable polymer wastes into the environment
everywhere from farmland to deep sea;1,2 and these wastes are threatening
biodiversity.3 Moreover, the vast increase of the petroleum consumption
relating to the economic growth of developing countries is threatening the
future supply of the resource. Recently, global warming has become a pressing
international issue; and incineration of the polymer wastes, though it has
advantages on waste reduction, leads directly to the increase of carbon dioxide.
In these situations, it is reasonable that polymers which undergo biodegrada-
tion and/or are produced from renewable resources became the mainstream of
environmentally benign polymers.
22
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polymers on your web browser, you should search many times with many words.
The situation will get worse because some websites use the term ‘plastics’ instead
of ‘polymer’. Among them, ‘green polymer’, though it is relatively obscure, is the
most useful in this chapter, because it includes both biodegradable polymers and
polymers prepared from biomass. In addition, the expression ‘environmental
benign polymer’ covers many types of polymers such as easily recyclable poly-
mers, very stable polymers without any eluted substances in water, and polymers
synthesized via atom-efficient (green) chemical processes, as well as biodegrad-
able polymers and polymers prepared from biomass.
In this chapter we describe ‘synthetic green polymers from renewable mono-
mers’ , including but not limited to biodegradable polymers. In the past, synthetic
green polymers attracted attention mainly as biodegradable polymers which
would resolve the problems of plastic waste disposal.5–9 Most of them are
aliphatic polyesters, and some of them are synthesized from petroleum chemicals.
On the other hand, the involvement of greenhouse gases with the global warming
since middle of the twentieth century has been suspected, and the increase
of greenhouse gases has been attributed to human activity including
the increasing use of fossil resources. On 11 December 1997, the Kyoto Protocol
was adopted by Conference of the Parties COP 3 held at Kyoto in Japan under the
United Nations Framework Convention on Climate Change (UNFCCC).10 In
the protocol, countries should reduce greenhouse gas emissions at the average
emission in 2008–2012 by the reduction rate of 5.2% from the 1990 level of
collective emissions of the world. After the enforcement of the protocol, emis-
sions of carbon dioxide became the major part of the environmental issues, and
many countries started to act against carbon dioxide emissions. One of the
prominent concepts for reducing carbon dioxide emissions is ‘carbon neutrality’,
contemplating the carbon atom’s cycle only on the surface of the earth.11,12
According to the ‘carbon neutrality’ concept, resources that have their origin
in products of photosynthesis are synthesized from carbon dioxide and their
use is not counted as carbon dioxide emission.13,14 Under this concept,
although people should avoid overspending forestry resources without plant-
ing, much fossil fuel use can be covered by renewable resources, achieving
reduction of carbon dioxide emission. The substitution from fossil fuel
resources to renewable fuel resources may be easy, e.g. most gasoline can be
substituted by ethanol and most fossil diesel can be substituted by biodiesel.
The substitution for polymer materials, however, is not so simple because of the
necessity of polymers with a variety of physical properties in industrial appli-
cations and therefore the necessity of a variety of resources for polymer pro-
duction. If all the polymers used in the world were limited to polyethylene,
polypropylene, and polystyrene, the situation would be similar to that of fuels;
but in fact it is not the case.
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24 Chapter 2
We should remind the reader that polymers were originally synthesized from
natural products. In ancient times, materials were made of substances available
without any chemical processing: e.g. branches, trunks, and leaves from trees,
and skins, hair, and bones from animals were used as they were. Since many
chemicals began to be industrially produced in the second industrial revolution
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
(later half of the nineteenth century), natural polymers have been chemically
modified to yield ‘naturally derived’ polymer materials for industrial applica-
tions. Though there may be some discussion, vulcanized natural rubber is the
first cross-linked polymer that was produced and used industrially, and cellu-
lose nitrate plasticized with camphor was the first thermoplastic polymer.15
Polymer materials derived from natural polymers, such as regenerated cellulose
and cellulose acetate, were developed late in the nineteenth century. While most
polymer materials were made from natural polymers in the nineteenth century,
synthetic polymers made from petroleum have dominated in the twentieth
century with the greatly development of petrochemical industries. Naturally
derived monomers (excluding monomers of natural polymers) had not attrac-
ted much attention in the polymer industry, perhaps because many of them
needed costly extraction processes from plants, animals, and microorganisms,
while substitutes obtained from petroleum have been available at a lower cost.
However, an air of fragility of sustainable petroleum supply (and the consequent
increase of petroleum price) and the recent developments of chemical engineering
and biotechnology for effective monomer production and purification have
accelerated research on synthetic polymers containing naturally derived mono-
mers (so-called renewable monomers) with the ‘carbon neutral’ concept.16,17
Table 2.1 summarizes the classification of renewable monomers. In this chapter,
renewable monomers that are used or have the potential to be used for polymer
syntheses and examples of their polymerization strategy are reviewed.
Direct extraction from Castor oil p-Coumaric acid (4- Triglycerides containing unsaturated a-Phellandrene
plants Sucrose Hydroxycinnamic bonds (e.g. tung oil, linseed oil, a-Terpinene
Malic acid acid) soybean oil) Rosin
Tartaric acid Vaniline Limonene
Fumaric acid Vanillic acid Pinene
Citric acid Gallic acid Phellandrene
Glycolic acid Eugenol Myrcene
Terpin (p-menthane- Cardanol Cinnamaldehyde
1,8-diol) Urushiol Cinnamic acid
p-Menthane-3,8-diol Curcumin Eugenol
Cineol Cardanol
Mandelic acid Urushiol
Chemical conversion Epoxidized triglycer- b-Malolactonate Fatty acids containing unsaturated Furfural
of plant extracts ides esters bonds (e.g. oleic acid, linoleic acid, Furfuryl alcohol
Synthetic Green Polymers from Renewable Monomers
26
Table 2.1 (Continued )
For Diels-Alder
For condensation polymers For addition polymers reaction
Collection from Cholesterol and its Peptides (protein Animal triglycerides and fatty acids
animals (some via derivatives digests) containing unsaturated bonds (e.g.
chemical Lactose Amino acids fish oil, docosahexaenoic acid)
conversion)
Chapter 2
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Triglycerides 10:0 12:0 14:0 16:0 18:0 18:1 18:2 18:3 20:0
HO HO 1
1
12
HO 1 HO 1
HO 12
12
15
28 Chapter 2
reactions is a well-known reaction sequence involving drying oils, accelerated
by driers such as cobalt, lead, and zirconium 2-ethylhexanoates. The reaction
has been used for a long time to form resins for coatings, paintings, flexible
films, binders for composite materials, and so on.19,21–24 The initial reaction
step of autooxidation is the abstraction of the hydrogen radical from methylene
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
O
O O O
O O
O
O
O O
O O
O O
O O
O
Plant oil O
Maleated derivative
HO
O (anhydride functional)
O OH
O HO
O
O O
O O
OH
O O O HO
O O
O O OH
Epoxidized derivative Polyol derivative
O O
O
O O
HO
O OH OH
HO O HO
O O
O O
O HO O
O O HO O HO
O O O O
O O O
HO O
O O
O
Maleated derivative Acrylated derivative
(acid functional)
Hydrolysis OH
Castor oil CH 3 (CH 2)5 CH CH 2 CH CH (CH 2)7 COOH
Ricinoleic acid
OH OH
CH 3 (CH 2)5 CH (CH 2 )10 COOH CH 3 (CH 2)5 CH CH 3 +
12-Hydroxystearic acid
OH
HOOC (CH 2)8 COOH
CH 3 (CH 2 )5 CH CH 2 CH CH (CH 2 )7 COOCH 3
Sebacic acid
Pyrolysis
Hydrolysis
CH 3 (CH 2 )5 CHO + CH 2 CH (CH 2 )8 COOCH 3 CH 2 CH (CH 2)8 COOH
10-Undecenoic acid
Addition of HBr
(in the presence of peroxide)
NH 3
Br CH 2 CH 2 (CH 2)8 COOH H 2 N (CH 2 )10 COOH
11-Aminoundecanoic acid
Figure 2.2 Strategies for synthesis of reactive monomers derived from castor oil.
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30 Chapter 2
and Diels–Alder reaction is that these reactions occur without a catalyst. Shi-
bata and his co-workers35,36 used bismaleimide as a cross-linker to yield tung
oil-based and dehydrated castor oil-based thermosets. In the polymerization
process, ene reaction, Diels–Alder reaction, and radical reaction occurred in
parallel. Çaylı and Küsefoğlu37 reported polymerization of linseed oil with
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
phenolic resin via ene reaction in the presence of a small amount of maleic acid.
Ene reaction of soybean oil with p-dinitrosobenzene (DNB) were reported by
Mutlu and Küsefoğlu.38 The polymeric form of DNB was prepared and reacted
with soybean oil via ene reaction to give hydroxylamine (–N–O–H) and
subsequently azomethine (–C¼N–) groups at cross-linking sites. Recently,
ultraviolet (UV)-curing of triglycerides via thiol-ene reaction has been reported.
Thiol-ene reaction is a free radically initiated reaction of thiol adding to
unsaturated C¼C bonds. Thiol-ene reaction of fatty acid was investigated by
Samuelsson et al.39 They reported the importance of trans unsaturated C¼C
bonds in the reaction. Chen et al.40 prepared a multifunctional thiol monomer
and a multifunctional allyl monomer from epoxidized soybean oil (ESBO).
The mixture underwent photopolymerization to form coating films. Black and
Rawlins41 synthesized allyl, acrylate, and vinyl ether derivatives of castor oil
and photopolymerized them with a multifunctional thiol compound, tri-
methylolpropane tris(3-mercaptopropionate), in the presence of photoinitiator.
Physical properties of cured films increased by aging for a week after UV irra-
diation. Ene reaction and Diels–Alder reaction are also used for modification of
triglycerides with reactive groups. Eren et al.42 reacted soybean oil with maleic
anhydride, and obtained a multifunctional anhydride compound. Reaction of
the anhydride compound with polyols gave cross-linked polyesters, and the
products were soft solids. Larock and his co-workers43,44 used Dilulins, which is
a compound derived from linseed oil and cyclopentadiene via Diels–Alder
reaction and ene reaction. They polymerized Dilulins with dicyclopentadiene
(DCPD) by cationic polymerization using boron trifluoride diethyl etherate,43 or
by ring-opening metathesis polymerization using Grubbs’ second generation
catalyst.44 DCPD content influenced physical properties of the thermosets.
Usual polymerization techniques for vinyl polymers, such as radical poly-
merization and cationic polymerization, have been often applied to cross-
linking of triglycerides and their derivatives. Commercial vinyl monomers are
usually added as a diluent to improve properties. In 1940s and 1950s, several
researchers reported ‘styrenated oils’ as copolymers of styrene and triglycerides.
Hewitt and Armitage,45 Schroeder and Terrill,46 Hoogsteen et al.,47
Boelhouwer et al.,48 and Crofts49 investigated the preparation of copolymers of
styrene and triglycerides, mainly using drying oils. This type of copolymers has
been revisited since 1990s by two research groups – Yagci and his co-workers
and Larock and his co-workers. Yagci and his co-workers50–52 devised a
macroinitiator method and a macromonomer method. In the macroinitiator
method, they synthesized a glyceride-based azo initiator by reacting 4,4 0 -azo-
bis(4-cyanopenanoyl chloride) with partial glycerides which is prepared by
glycerolysis of triglycerides.50 The glyceride-based azo initiator was reacted
with styrene to yield a clear solid after monomer distillation in vacuum. In the
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32 Chapter 2
As described above, triglycerides polyol are derived by several methods.
Polyurethanes can be obtained when triglyceride polyols are reacted with
multifunctional isocyanate compounds. Numerous reports on this type of
polyurethane are found, and some of them involve polyurethane foam. In
1959, castor oil-derived polyurethane foams were prepared by Yeadon et al.66
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
34 Chapter 2
proceeded to yield copolymers. The molecular weight of copolymers decreased
with an increase in the feed ratio of ricinoleic acid lactones. Poly(ricinoleic acid)
with high molecular weight (up to Mw ¼ 98 000) was successfully synthesized by
Ebata et al.95 via lipase-catalyzed polymerization of methyl ricinoleate.
The obtained poly(ricinoleic acid) was a viscous liquid with the Tg of –74.8 1C.
Domb and Nudelman96 synthesized ricinoleic acid-based polyanhydride from
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
derivatives are also included in this section, though most of them are not volatile
(and so are not called essential oils). Some other phenolic compounds, most of
which are derived from the shikimic acid pathway, are also extracted from plants.
2.3.1 Terpenoids
Homopolymerization of terpenoids using C¼C bonds is not as easy as
homopolymerization of industrially used vinyl monomers. The trial study on
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36 Chapter 2
the polymerization of terpenes was reported by Carmody and Carmody in
1937.103 They tried to polymerize pinene by aluminum chloride and obtained
the mixture of an oily polymer product and a hard polymer product. Roberts
and Day104 also tried to polymerize a-pinene, b-pinene, and limonene by sev-
eral kinds of Lewis acid catalysts in 1950. The approximate molecular weights
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
of the polymer obtained from a-pinene, b-pinene, and limonene were reported
as 700, 3100, and 1200, respectively. Emulsion polymerization of myrcene was
studied by Johanson et al.105 in 1948. The obtained polymers were low in
mechanical properties, and the tercopolymer from myrcene, styrene, and
butadiene showed good tensile properties. Marvel and Hwa106 reported poly-
merization of myrcene with triisobutylaluminum and vanadium trichloride.
Inherent viscosities of the products are 2 to 5.5 dL g1. Cawse et al.107 syn-
thesized hydroxyl-terminated polymyrcene using hydrogen peroxide-initiated
polymerization, and the number-average molecular weight (Mn) of the polymer
was up to 4000. In 1992 Keszler and Kennedy108 reported synthesis of high
molecular weight poly(b-pinene) by ‘H2O’/EtAlCl2 system at 80 1C (up to
Mn ¼ 39 900); and Sawamoto and his co-workers109 reported cationic poly-
merization of a-pinene and b-pinene with the AlCl3/SnCl3 binary catalyst in
1996 (up to Mn ¼ 6400). Subsequently Sawamoto and his co-workers110
achieved the living cationic isomerization polymerization of b-pinene using an
initiating system with HCl-2-chloroethyl vinyl ether adduct and TiCl3(OiPr) in
1997. Though Mn of the polymer products was less than 5 103, the poly-
merization system opened the synthetic route to block copolymerization and
end functionalization.111–113 Guiné and Castro114 polymerized b-pinene with
C2H5AlCl2 (up to Mw ¼ 2430) and determined the Mark-Houwink-Sakurada
constants. More recently, the polymerization to obtain high molecular weight
poly(b-pinene) (up to Mn ¼ 25 100) was achieved by Satoh et al.115 in 2006,
using EtAlCl2, Et3Al2Cl3, and AlCl3 as a Lewis acid and a 1:1 mixture solvent
of CH2Cl2 and methylcyclohexane. After hydrogenation reaction, the Tg
of the polymer was found to increase to 130 1C, and the polymer has
good transparency and thermal properties comparable to polystyrene
and poly(methyl methacrylate). They also achieved the polymerization of
a-phellandrene. Figure 2.4 shows the mechanisms of cationic polymerizations
of b-pinene and a-phellandrene. A polymer synthesized from a myrcene
derivative was reported by Hillmyer and his co-workers,116 where a cyclic diene
3-methylenecyclopentene prepared by ring-closing metathesis of myrcene was
polymerized by living cationic polymerization using i-BuOCH(Cl)Me/ZnCl2
system (up to Mw ¼ 22 000) (Figure 2.5). Radio frequency plasma poly-
merization of terpinen-4-ol, the major component of tea tree oil, was reported
by Bazaka and Jacob.117 They obtained the smooth thin film on a glass
substrate. The structure of the polymer may differ from the repetition of the
monomer because of branching and cross-linking.
Research on the copolymerization of terpenoids with industrially used vinyl
monomers or other monomers have been often reported. Littmann118 intro-
duced terpene-maleic anhydride resins for industrial use in alkyd resins in
1936. The resin was synthesized from a-terpinene and maleic anhydride via
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i-BuOCH(Cl)Me
ZnCl2
CH3 H3C
N N
CPD CH3
H3C CH3
38 Chapter 2
and vinyl monomers. They reported many copolymers: geraniol-co-styrene,129
a-terpineol-co-methylmethacrylate (MMA),130 citronellol-co-vinyl acetate,131
limonene-co-MMA,132 citronellol-co-acrylonitrile (AN),133 a-terpineol-co-
styrene,134 a-terpineol-co-butylmethacrylate,135 linalool-co-vinyl acetate,136
limonene-co-styrene,137 and geraniol-co-MMA.138 They found that the poly-
(geraniol-co-MMA) was a liquid crystalline polymer.138
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
CO 2Et OH CO 2 Et
OH 1) PBr 3 1) mCPBA
CO 2Et CO 2Et
2) NaCH(CO 2Et)2 2) LDA
R2
O O R4
R2
N
3 O
+ R Zn
N O
R1 O
O
R1
O
+ CO 2 R 1, R 2 = Et or iPr
R 3 = H, R 4 = CH 3 or CF 3
n
resins which can react with epoxy resins, bisphenol A-based epoxy resin, and
epoxidized soybean oil, to give coating films. The films were found to be hard,
glossy, and brittle when using the bisphenol A-based epoxy resin. Many types
of terpene-based reactive monomers are available from a chemical company,
Yasuhara Chemical Co., Ltd. (Fuchu, Hiroshima, Japan). Kimura et al.146
prepared the terpenediphenol-based benzoxiazine and cured it with epoxy resin.
The benzoxiazine ring opens when heated and formed phenolic hydroxyl
groups which can react with epoxy groups. The cured resin showed good
properties in thermal stability, mechanical properties, electrical insulation, and
water resistance, compared with the cured resin from bisphenol A type resin.
Xu et al.147 synthesized an epoxy resin from 1-naphthol, limonene, paraf-
ormaldehyde, and epichlorohydrin (Figure 2.8). The epoxy resin was reacted
with dicyandiamide or bisphenol A-formaldehyde novolac resin. The cured
polymers had a high glass transition temperature, high thermal stability, and
good water resistance compared with bisphenol A type epoxy resin. Wu et al.148
synthesized an terpinene-based epoxy resin from the hydrogenated terpinene-
maleic anhydride and epichlorohydrin. Subsequently they prepared polyols
from the epoxy resin, and cured the polyols with a 1,6-hexamethylene diiso-
cyanate derivative to give polyurethanes with high impact strength, high flex-
ibility, and high thermal stability.149 Shibata and his co-workers150 reported
organic-inorganic hybrid nanocomposites synthesized from terpene-based
acrylate resin and methacryl-substituted polysilsesquioxane. They prepared
the transparent hybrid nanocomposites by photocuring the acrylate and the
OH
OH OH
2
(CH 2O)n
OH OH
p-toluenesulfonic
BF 3·Et2O acid
CH 2
n
CH2
CH2 CH
O
O O
CH2
CH2 CH CH Cl CH2 CH
2 O
O
NaOH
CH2
n
40 Chapter 2
methacrylate groups in the presence of a radical photoinitiator. The mechanical
properties of cured terpene-based acrylate resins were improved by
polysilsesquioxane.
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
2.3.2 Phenylpropanoids
Phenylpropanoids are biologically synthesized from phenylalanine as described
above. Among them, cinnamic acid is synthesized directly from phenylalanine
by phenylalanine ammonia-liase (PAL), and p-hydroxycinnamic acid (p-
coumaric acid) is synthesized from cinnamic acid by cinnamic acid 4-hydroxylase
(C4H, an enzyme in the cytochrome P-450 family).102 The phenylpropanoid
metabolic pathway is important for plants to synthesize lignin, and some
phenylpropanoids are seen at junctions of cell wall polysaccharides such as
hemicellulose and pectin.151
Phenylpropanoids having a cinnamoyl group (Ph–CH¼CH–CO–), often
found in essential oils, are most usable monomers among phenylpropanoids.
Cinnamaldehyde, which is the main component of cinnamon oil, can be easily
transformed into cinnamic acid. Though the cinnamoyl group has a C¼C
double bond, the successful addition homopolymerization has not been
reported, as far as we could find. The usual bulk polymerization of ethyl cin-
namate using a radical initiator gave a polymer with the molecular weight of
2300.152 On the other hand, its photodimerization reaction (2þ2 cycloaddition,
Figure 2.9) by irradiation of UV light (lB280 nm) is a well-known reaction
and often used for preparation of photocross-linked materials. Recently,
homopolymerization of cinnamyl groups (Ph–CH¼CH–CH2–), which also had
been known as an ‘unpolymerizable’ group for a long time, was reported by
Washburn et al.153 using the topological control process, in which cinnamyl
alcohol was absorbed onto a metallic substrate and subsequently photo-
polymerized at very low temperature (100 K) to yield very thin film of poly
(cinnamyl alcohol) with Mn 5.0 105. However, this polymerization process
is very particular and will not become popular for industrial production.
O
O
O
O
hν
+ [2+2]
O
O
O
O
42 Chapter 2
(a)
H O
C O
C CH
H HN NH HC
HC
CH Si O Si
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O O O H
HN O H
O C C
O N H
Si Si
H Si Si O
H N O
C C O
O O
H O HC
O
Si O Si HN CH
O
H
H N H O
C C N C
H H C
O H
(b) RO
O
RO OR O O
OR
OR
RO RO O
OR
R: H or
HC CH
deformed when heated at 50 1C, and they can recover their original shapes at
ambient temperatures when exposed to UV of a different wavelength.
Since photodimerization of cinnamoyl-carrying polymer using linearly
polarized light was reported by Schadt and his co-workers,173 anisotropically
photocross-linked cinnamate polymers have been known as a durable material
for liquid crystal alignment174 and have been studied by many researchers.175–182
Murase et al.183 reported the large refractive index change by photodimerization
of a cinnamate polymer and Nagata et al.184 proposed cinnamate polymers as a
material for holographic recording.
Coumarin and its derivatives, which are found in many plants, also show
photodimerization by irradiation of UV light with longer wavelength
(l ¼ 300B350 nm) than a cinnamoyl group. Trenor et al.185 wrote a detailed
View Online
Parmanent network
O O PPG O
O O O
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
Temporary network
O
O O
PEG
O PEG
PEG O
O PEG
O
O
review paper on coumarins in polymers. Coumarin and its derivatives are used
for functional groups in polymers.186–195 Polymers containing coumarin groups
are also used for photoalignment of liquid crystals.190–193 Some coumarin-
containing polymers were found to exhibit electroluminescence.188 Biopolymers
carrying coumarin groups were investigated by Yamamoto et al.194 and
Wondraczek et al.195 to obtain cross-linkable poly(Lys) and polysaccharide
sulfates, respectively.
Hydroxyl-functionalized cinnamic acid derivatives such as p-coumaric acid
(p-hydroxycinnamic acid), ferulic acid, and sinapinic acid are attractive
monomers for syntheses of high-performance polyesters. The obtained polye-
sters are also expected to be biodegradable in the case of copolymerization with
aliphatic hydroxy acids such as lactic acid. Tanaka et al.196 reported the ther-
mal polycondensation of p-coumaric acid at 550 1C without any catalyst under
high pressure up to 80 kbar (in the solid state) in 1975. They obtained red or
brownish-red hard solids insoluble in conventional organic solvents. Higashi
and his co-workers197 synthesized copolyesters of p-coumaric acid and 4-
hydroxybenzoic acid or their methoxy substitutions (ferulic acid, vanillic acid,
or syringic acid) by polycondensation using hexachlorocyclotri(phosphazene)
in pyridine in 1981. The obtained polymers that exhibited UV spectra different
View Online
44 Chapter 2
1
from those of monomers and inherent viscosity of 0.3 to 1.3 dL g . Palacios
and his co-workers reported syntheses of poly(p-coumaric acid)198 and poly-
(ferulic acid)199 using thionyl chloride in 1985. The melting temperatures of
poly(p-coumaric acid) and poly(p-ferulic acid) were 313 1C and 325 1C,
respectively. These polymers showed signs of thermotropic liquid crystallinity
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
a O
R1
O n
R1 = H or OMe
R1 R2
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
b
O O
R3
O m O n
R 1, R 2, R 3 = H or OMe
c
O O
O
CH
CH3
O m n
d
O O O
O
CH 2
O l O m n
e
O O
CH 3 CH 3
O O (CH2)10 O O
Si O Si
CH 2 CH 2
O CH 3 CH 3 O n
CH 3
f
O O O
O O
O
(CH 2)6
O O H H n
O
O
g
O O
(CH 2)6
O O O O
O (CH2)4 O n
H 3CO
CH 3 CH 3
2 HO
CH 3 CH 3 H 3CO Si O Si OCH3
H Si O Si H CH 3 CH 3
n
CH 3 CH 3 HO OH
n
46 Chapter 2
226
available from General Electric Co. Hagenaars et al. reported character-
ization of polycarbonates containing bisphenol A, eugenol, and siloxane oli-
gomer units. Waghmare et al.227,228 synthesized polyesters from phenylindane
bisphenol (or bisphenol A), diphenyl terephthalate, diphenyl isophthalate, and
the eugenol end-capped siloxane polymer. Masuda and his co-workers229
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
OH O O OH
H2O2
48 Chapter 2
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
Figure 2.15 Printed circuit board manufactured from lignin derivatives developed by
Hitachi, Ltd. Courtesy and reproduced with permission of Hitachi Ltd.,
Japan. r Hitachi Ltd, Japan.
OCH 3 OCH 3
O2
HOOC OH O
Laccase
CO 2 , H 2O
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
OCH 3 OCH 3 n
50 Chapter 2
(C 15 )
(a) OH
R:
R
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
(C 15 )
(b) OH
R:
OH
Figure 2.17 Structures of (a) cardanol and (b) urushiol (typical structures).
O O O
HO
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
Figure 2.18 Structures of (a) tulipalin A and (b) tulipalin B, in comparison with
(c) MMA.
52 Chapter 2
Multiple hydroxyl groups restrict usage of these carbohydrates as monomers
for linear polymers. Sachinvala et al.362 reported sucrose-based monomers
carrying unsaturated C¼C bonds for cross-linked polymers. Preparation of
sugar-based linear polymers are more challenging than cross-linked sugar-
based polymers. Researchers have found many ways to synthesize linear
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
polymers from sugars, but synthesized polymers have been somewhat biased
towards side-chain polymers (called as glycopolymers) mainly pursuing bio-
functionality.363 For example, 3-O-methacryloyl-1,2:5,6-diisopropylidene-D-
glucose (MDG) was synthesized from 1,2:5,6-diisopropylidene-D-glucose and
methacryloyl chloride, and polymerized using AIBN as radical initiator by
Kimura and Imoto.364 After polymerization, the isopropylidene protecting
groups were hydrolyzed to give poly(3-O-methacryloyl-D-glucose). From the
point of view of environmental polymeric materials, removing of protecting
groups is not necessary, even if it enhances the biocompatibility of the polymer
material. Al-Bagoury and Yaacoub365 investigated thermal, mechanical, and
rheological properties of copolymers of MDG and butyl acrylate prepared by
emulsion polymerization. The Tg of the copolymers varied from –54 1C to
167 1C depending on the composition of MDG to butyl acrylate. The Young’s
modulus increased with an increase of MDG contents, but the film became
more brittle. Most recently, Barros et al.366 adapted a selective Mitsunobu
reaction to synthesize a sucrose-based methacryl monomer (Figure 2.19). This
selective reaction is very attractive for synthesis of sugar-based polymers.
Preparation of cross-linked sucrose-based polymers by photopolymerization
was achieved by Acosta Ortiz et al.367 using thiol-ene reaction. They synthe-
sized sucrose allyl ether, mainly diallyl sucrose, and polymerized the sucrose-
based monomers with dithiothreitol by UV irradiation in the absence or
presence of a photoinitiator.
Condensation polymers containing sucrose or glucose units in the main chain
were also reported. Cross-linked polyurethanes prepared from polyethylene
glycol (PEG), diphenylmethane diisocyanate (MDI) and a sugar (glucose,
fructose, or sucrose), were reported by Zetterlund et al.368 The storage mod-
ulus, the Tg, and the stress at break increased with an increase of sucrose
content; and the strain at break decreased with an increase of sucrose content.
HO OH OH
O , DIAD, PPh3 O O OH
O O O
HO OH OH O
HO
O HO OH OH
DMF, r.t., 24 h HO
OH O
HO
HO OH
O
DIAD: diisopropyl azodicarboxylate
N O
O N
Figure 2.20 Structures of sugar diols: (a) D-isosorbide, (b) isomannide, (c) methyl
4,6-O-benzylidene-a-D-glucopyranoside, (d) methyl 2,6-di-O-pivaloyl-
a-D-glucopyranoside, (e) 1,2:5,6-di-O-isopropylidene-D-sorbitol, and
(f) 2,3,1 0 ,3 0 ,4 0 ,6 0 -hexa-O-acetylsucrose.
View Online
54 Chapter 2
synthesis of polyurethanes from methyl 4,6-O-benzylidene-a-D-glucopyranoside
and two diisocyanates, hexamethylene diisocyanate and methyl 2,6-diisocyana-
tohesanoate (lysine diisocyanate). They also synthesized another sugar diol,
1,2:5,6-di-O-isopropylidene-D-sorbitol (Figure 2.20e), as a monomer of
polyurethane. Jhurry and Deffieux388 synthesized a sugar diol from sucrose
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
O O
O O
(CH2)4 O OH
O
R = (CH 2 )6, (CH 2)8 , (CH 2)10 O
or CH2 –C 6 H 4–CH 2 HO OH OH O O
HO
O
R
HO O (CH2)4 O O
n
56 Chapter 2
a R= (CH 2)6
O O
O O
R CH 2 CH 2
O O
n CH 2 CH 2
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
b
O O
O O
(CH 2)2
O O
n
c
O O
O O
(CH 2)6
N N
H H d
n O O
O
e N N
H O H
O O H n
N
O
N
H
n
R= (CH 2)4
f CH 2
O CH
CH 2 CH 2
H O O H
N N O O O
R
CH 2 CH 2
O O
n
g h O
O
N CH 2
O O
O
n n
58 Chapter 2
disaccharide, D-glucopyranosyl-D-glucopyranoside, and it has two glucose
units bound at each C1 position. Trehalose is found in many organisms, such as
bacteria, fungi, plants, and invertebrates including insects. Trehalose is known
to play an important role in cryptobiosis of many organisms. Recently, an
enzymatic process for the industrial synthesis of trehalose was established by
Hayashibara Co. Ltd,434 and trehalose became available at a low cost (o$3 per
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
1 kg). Both erythritol and trehalose have two primary hydroxyl groups and
several secondary hydroxyl groups. The difference in reactivity between pri-
mary hydroxyl groups and secondary hydroxyl groups is applicable to regio-
selective reaction which enable facile synthesis of linear polymers from
erythritol or trehalose. Examples of regioselective reactions are acetalization
with benzaldehyde derivatives, iodine substitution, and enzymatic esterifica-
tion. Polymers synthesized from trehalose are reviewed in detail elsewhere.435
Research reports on polymers from erythritol are very few, and most of
them are related to polyester. Uyama et al.436 and Hu et al.437 reported
polymerization of sugar alcohols with divinyl sebacate and adipic acid,
respectively, using lipase. Barrett et al.438 reported the thermal polycondensa-
tion of erythritol with dicarboxylic acids to obtain cross-linked elastomeric
materials. The regioselective esterification using rare-earth trifluoro-
methanesulfonate, reported by Takasu et al.,439 will be a convenient method
for preparation of linear polymer from erythritol or glucaric acid. 1,4-Anhy-
droerythritol is a cyclic bifunctional compound derived from erythritol. This will
be used as a monomer for polycondensation. Imai et al.440 reported the
ring-opening polymerization of 1,4-anhydroerythritol to obtain hyperbranched
carbohydrate polymers, and Wachenfeld and Burchard441 synthesized epoxy
resins from 1,4-anhydroerythritol and bisphenol A diglycidyl ether. Bachmann
and Thiem442 transformed 1,4-anhydroerythritol into 2,3-diamino-2,3-dideoxy-
1,4-anhydroerythritol, and polymerized the resulting diamine with aro-
matic and aliphatic dicarboxylic acid dichlorides to obtain polyamides
(Figure 2.24).
Glucaric acid is a compound having two carboxyl groups and four secondary
hydroxyl groups. It can be obtained by oxidation of glucose, and recently
Moon et al.443 constructed a recombinant E. coli that produces glucaric acid.
Polyamides based on glucaric acid were synthesized by Kiely et al.444 via simple
condensation reactions of esterified glucaric acid with diamines (Figure 2.25).
Interestingly, even an aromatic compound with a benzene ring is also found
to be synthesized by fermentation. Horinouchi and his co-workers445 reported
that two genes, griI and griH, conferred the ability of in vivo production of
3-amino-4-hydroxybenzoic acid (3,4-AHBA) on E. coli (Figure 2.26). This
pathway is independent of the shikimate pathway, which is a well-known
pathway for production of aromatic amino acids in bacteria, fungi, algae, and
higher plants. Homopolymerization of 3,4-AHBA yields polybenzoxazole
(ABPBO) (Figure 2.27), which shows the liquid-crystalline phase during
polymerization.446–449 In the near future, high-performance industrial plastics
from renewable monomers may appear.
Monomers that can be obtained by fermentation are shown in Figure 2.28.
View Online
H 2N NH 2 H H
N N R
Cl R Cl
+
− HCl O O
O O O
O n
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
R= (CH 2 )8
(CH 2)10
OMe
O OH OH
HO
O H H
+ H 2N R NH 2 N N
HO O R
OH OH O
n
OH
Methyl D -glucarate
1,4-lactone
R= (CH 2)2
CH 2 CH 2
CH 2 CH 2
(CH 2)4
(CH 2)12 CH 3
Figure 2.25 Synthesis of polyamides from esterified glucaric acid diamine and
dicarboxylic acid dichloride.
OPO 3 H 2
OPO 3 H 2
NH 2
HOOC NH 2 HOOC O HOOC NH 2
O
+
GriI GriH
CHO OH OH
OH
OH
60 Chapter 2
HOOC NH 2 N
Polyphosphoric acid O
OH n
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00022
(i) O (j)
OH
OH HOOC NH 2
HO
OH
HO
HO OH
O
many processes for its production, it may become improved in the decade ahead.
Alternatively, even if a monomer is now derived only from petroleum,
it may become a renewable compound in the decade ahead.
Most current technology is beginning to change the view of renewable
polymers; and nowadays ethylene and propylene are becoming renewable
monomers, because ethylene and propylene can be derived from ethanol, which
is a renewable compound.450–452 Furthermore, Bio-PET, which is synthesized
using ethylene glycol produced from sugar cane, will start to be used in motor
vehicles from the year 2011.453 In addition, synthesis of terephthalic acid from
p-cymene (an essential oil) or limonene has been reported.454 Possibly, these
technologies would greatly change the present concept of green polymers,
because current commodity polymers become green polymers. However, there
is no assurance that the day will come soon. At least now, we should look
towards naturally occurring compounds and struggle to find many green
polymers that covers our livelihood. Hundreds varieties of renewable mono-
mers will offer the solutions.
This review was written without an emphasis on biodegradability. Green
polymers from renewable resources are not necessarily biodegradable. From
the view of carbon neutrality, it is often said that carbon dioxide is released by
biodegradation of polymers as well as by incineration of polymers. However, I
do not wish to downplay the importance of the biodegradability of green
polymers. I would rather emphasize the importance of biodegradability, by
considering differences in the releasing rate of carbon dioxide: biodegradation
is a slow reaction, while incineration is a rapid reaction. Though it might be a
misleading expression, we can fix carbon dioxide as a form of biodegradable
polymers. Of course, the amount should be limited to avoid overspending
resources. At least biodegradable polymers can prevent the environmental
pollution with nondegradable polymers which are unconsciously discarded.
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Polyhydroxyalkanoates: The
Emerging New Green Polymers
of Choice
RANJANA RAI AND IPSITA ROY
3.1 Introduction
Synthetic plastics are imposing serious threats to our environment. Since their
introduction in the 1950s plastics have become an absolute necessity of our life.
However these plastics are not degradable. Non-biodegradable plastics accu-
mulate at the rate of 25 million tons per year and have therefore become one of
the main environmental hazards. This problem is compounded with the fact
that the resources for crude oil is also decreasing. Therefore, in the present
scenario when global warming, climate change and dwindling fossil carbon
resources have taken centre stage, scientists from all over the world are looking
for greener eco-friendly alternatives to petrochemical-derived plastics. This
alternative has come in the form of biodegradable plastics and one such family
attracting considerable interest is the polyhydroxyalkanoates, PHAs. Poly-
hydroxyalkanoates are linear polyesters of 3, 4, 5 and 6-hydroxyalkanoic acids
accumulated by numerous Gram positive and Gram negative bacteria through
the fermentation of sugars, lipids, alkanes, alkenes and alkanoic acids (Figure
3.1). These are, therefore, bio-based polymers and once extracted exhibit
thermoplastic and elastomeric properties closely resembling synthetic plastics.
79
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80 Chapter 3
In fact, the properties of these polymers can be tailored by controlling the
carbon feed. They are recyclable and degrade into CO2 and water. In addition,
these polymers are also biocompatible. It is because of these reasons that PHAs
are becoming increasingly popular as environmentally friendly materials with
industrial, agricultural and medical applications.1–3
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R1 O R2 O
CH3 CH3
O O O
x x n
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Depending on the number of carbon atoms in the monomeric unit, PHAs are
classified as short-chain length PHAs, scl-PHAs, that contain 3–5 carbon
atoms, examples are poly(3-hydroxybutyrate), P(3HB), poly(4-hydroxy-
butyrate), P(4HB), and medium-chain length PHAs, mcl-PHAs, that contain
6–14 carbon atoms, examples are poly(3-hydroxyhexanoate), P(3HHx), and
poly(3-hydroxyoctanoate), P(3HO). Also depending on the kind of monomer
present, PHAs can be a homopolymer containing only one type of hydroxy-
alkanoate as the monomer unit, e.g. P(3HB), P(3HHx), or a heteropolymer
containing more then one kind of hydroxyalkanoate as monomer units, e.g.
poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV), and poly-3-
hydroxybutyrate-co-3-hydroxyhexanoate, P(3HB-co-3HHx).14,15 Mcl-PHAs
are more structurally diverse than scl-PHAs. Here, the ‘R’ group can vary from
propyl to tridecyl (e.g. 3-hydroxyhexanoate), may contain an aromatic group
(e.g. mcl-PHA with para-methylphenoxy and meta-methylphenoxy) and the
alkyl side-chain can be saturated (e.g. 3-hydroxyoctanoate) and unsaturated,
e.g. 4- hexenoic, 3-hydroxy-8-nonynoate and 3-hydroxy-10-undecynoate.16,18
Some mcl-PHAs have also been found with the hydroxyl group on the C-2, C-4,
C-5 and C-6 carbon atoms. In mcl-PHAs, the presence of functional groups like
halogen, carboxyl, hydroxyl, epoxy, phenoxy, cyanophenoxy and nitrophenoxy
are particularly important, as they allow further chemical modifications of
these PHAs, leading to the production of novel biomaterials with tailorable
properties. At the time of writing, more then 150 units of PHA monomers have
been reported (Table 3.1 and Figure 3.2).
Table 3.1 The broad spectrum of monomers found in PHAs (adapted from Zinn et al.28). 82
3-Hydroxy 3-Hydroxy acid 3-Hydroxy acid 3-Hydroxy acid Other than Other functional
acid (unsaturated) (branched) (substituted side chain)3-hydroxy acid Aromatic side chain groups
3-Hydroxyacids
HO O HO O
OH OH
O H3C
H3C O
HO O
OH
H3C O
3-Hydroxyoctanoate
II. 3-Hydroxyacids(Unsaturated)
HO O HO O
OH OH
H3C O H3C O
4-Hexenoic 5,8-Tetradecadenoic
OH O HO O
H3C OH
H3C O OH
O O
O H3C
8-Methylnonanoic 2,6-Dimethyl-5-heptenoic
HO O HO O
OH OH
F O F O
7-Fluroheptanoic 7-Cyanoheptanoic
2-Hydroxydodecanoate 4-Hydroxyhexanoate
84 Chapter 3
3-Hydroxy-7-oxooctanoate 8-Acetoxy-3-hydroxyoctanoate
therefore it is securely available to the organism for future use.29,31 The bio-
synthesis of PHAs involves two major steps – the first step leads to the gen-
eration of hydroxyacyl-CoA substrates and the second leads to the
polymerization of these substrates into PHAs. This polymerization of PHAs is
carried out by the enzyme called PHA synthase encoded by the phaC gene
(Figure 3.4).32
Three metabolic pathways (Pathway I: Chain elongation reaction; Pathway II:
Fatty acid b-oxidation; Pathway III: Fatty acid de novo biosynthesis) are used by
organisms to produce the hydroxyacyl-CoA substrates (HACoA), which are then
polymerized into PHAs (Figure 3.4). Pathway I is the best known among the PHA
View Online
O OH O R O
CH3
CH3 CH3
O + R SCoA CH3 O
n n+1
Figure 3.5 Metabolic pathways for the production of PHAs (adapted from Kim
et al.38).
biosynthetic pathways and is found in Cupriavidus necator.29,33 Here, condensa-
tion of two acetyl-CoA molecules from the tricarboxylic acid (TCA) cycle takes
place to form acetoacetyl-CoA.34,36 Acetoacetyl-CoA is then converted to (R)-3-
hydroxybutyryl-CoA by the (R)-specific acetoacetyl-CoA reductase (phaB).
Finally, the PHA synthase enzyme catalyses the polymerization via esterification of
3-hydroxybutyryl-CoA into poly(3-hydroxybutyrate), P(3HB).36 Pathway II, the
fatty acid b-oxidation-involving pathway, was deciphered when fluorescent
Pseudomonads, such as Pseudomonas oleovorans, Pseudomonas putida and Pseu-
domonas aeruginosa, were found to accumulate PHA consisting of mcl-(R)-3HA
units from alkanes, alkanols, alkanoates and oils.33,37 Here, the fatty acids are first
converted to the corresponding acyl-CoA thioesters which are then oxidized by
fatty acid b-oxidation via trans-2-enoyl-CoA and (S)-3-hydroxyacyl-CoA to form
3-ketoacyl-CoA. 3-ketoacyl-CoA is then cleaved by a b-ketothiolase to form
acetyl-CoA and an acyl-CoA comprising of two fewer carbon atoms
compared to the acyl-CoA that entered the first cycle. The trans-2-enoyl-CoA, (S)-
3-hydroxyacyl-CoA and 3-ketoacyl-CoA intermediates formed are utilized for the
generation of (R)-3-hydroxyacyl-CoA derivatives which are then polymerized to
form mcl-PHAs. Fatty acid de novo biosynthesis is involved in Pathway III and is of
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86 Chapter 3
significant interest because it helps generate PHA monomers from structurally
unrelated, simple, inexpensive carbon sources such as glucose, sucrose and fructose
(Figure 3.5). In this pathway, the enzyme acyl-CoA-ACP transferase (encoded by
phaG) transfers the hydroxyacyl moiety from (R)-3-hydroxy-acyl carrier protein to
coenzyme A, thus forming (R)-3-hydroxyacyl-CoA, which acts as the substrate for
the PHA synthase enzyme.36,38
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88 Chapter 3
Table 3.3 Mechanical properties of various PHAs and conventional plastics
(adapted from Castilho et al.67).
Elongation
Polymer Tensile strength Modulus to break (%) Reference
90 Chapter 3
from the families Pseudonocardiaceae, Micromonosporaceae, Thermomono-
sporaceae, Streptosporangiaceae and Streptomycetaceae predominantly degrade
P(3HB) in the environment. These microbes secrete extracellular enzymes that
solubilize the polymer and these soluble products are then absorbed through their
cell walls and utilized. Some PHA-producing bacteria are able to degrade the
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can degrade in septic tank systems and this would include hygienic wipes and
tampon applicators. They can also be used to manufacture medical surgical
garments, upholstery, carpet, packaging, compostable bags and lids or tubs for
thermoformed articles. PHA-based latex can be used for making water-resistant
surfaces to cover paper and cardboard.36,86
92 Chapter 3
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Figure 3.6 (a) P(3HO) scaffold frontal view, 18 mm in diameter and 20 mm in length.
Three leaflets of porous P(3HO) were sutured into the conduit proximal
view. (b) Gross appearance of tissue-engineered seeded conduit 24 weeks
in vivo (distal view). Clear separation of all three leaflets from the conduit
wall is shown. (c) Gross appearance of tissue-engineered unseeded conduit
4 weeks in vivo (proximal view).89
Using this technique, P(3HO) and P(4HB) could be moulded into a complete
3-leaflet valve scaffold without the need for suturing.88,90 Novel hybrid valves
which were fabricated from decellularized porcine aortic valves and coated with
P(3HB-co-3HHx) were also developed. The results in vivo indicated that the
P(3HB-co-3HHx) coating reduced calcification and promoted the repopulation
of the hybrid valve with the recipient’s cells, resembling native valve tissue.91
PHAs like P(3HB), P(3HB-co-3HHx) and blends of PHA with PDLLA have
also been used as biomaterials for nerve regeneration. One of the early studies
on P(3HB-co-3HHx) as conduit material for nerve regeneration was carried out
by Yang et al.92 in 2002. This study showed that the foetal mouse cerebral
cortex cells were able to grow well when seeded on P(3HB-co-3HHx) films.92
Similarly PHAs have also been extensively explored for hard tissue engineering.
P(3HB) and composites of P(3HB) with bioactive nanobioglass (n-BG) 45S5
have been extensively studied for bone tissue engineering. The composite P(3HB)/
n-BG showed good biocompatibility with the seeded MG-63 human osteoblast cell
line. Incorporation of vitamin E in the composite further enabled better attach-
ment, proliferation and differentiation of the osteoblast cells.93–95 Composites of
mcl-PHA, P(3HO)/n-BG two-dimensional (2D) scaffold or film have also been
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Figure 3.7 Scanning electron micrographs of rabbit bone marrow cells seeded on
PHBHHx scaffold after 10 days of incubation (1000). (a) Cell clumps;
(b) round cells with fibrillar collagen (F,C) attached by filapodia; (c) cells
with extracellular matrix (M) and calcified globuli (G).97
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94 Chapter 3
allowed chondrocytes to express and produce type II collagen more than the
P(3HB) only scaffold (control). The P(3HB-co-3HHx) component in the blend
P(3HB)/P(3HB-co-3HHX) scaffold provided better surface properties for
anchoring type II collagen filaments and their penetration into internal layers
of the scaffolds. These results suggested that the cells underwent chondrogenic
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higher than the cost of the gasoline products in the Chinese market (US$800 per
ton).106 Carbon dioxide emitted from burning fossil fuel is one of the main con-
tributors to greenhouse gases, GHG. In fact in the United States carbon dioxide
released from fossil fuel combustion represents 98% of the total GHG emission.
Studies were carried out by Yu et al.107 on the GHG emissions and fossil energy
requirement per kg of bioplastics produced. Three PHA polymers from different
carbon sources PHA-G (glucose), PHA-O (vegetable oil) and PHA-BS( black
syrup) were studied. The analysis indicates that PHA bioplastics can reduce GHG
emissions with only 0.49 kg CO2-e being emitted from production of 1 kg of resin.
Compared with 2–3kg CO2-e of petrochemical counterparts, PHAs have the
potential of about 80% reduction in the CO2-e. Interestingly, other bio-based
polyesters such as polylactide, PLA, showed high GHG emission, 1.8 kg CO2-e.
The total fossil energy requirement per kg of PHAs was between 40 and 59 MJ
kg1, again lower than those of the petrochemical counterparts (78–88 MJ kg1
resin). Figure 3.8 compares the GHG emissions and fossil energy requirements of
representative petroleum and bio-based polymers.107 Although the energy of
combustion of the PHA biofuels was lower and the estimated cost of production
higher, it has however opened a new avenue for PHA application. The significance
of PHA-based biofuels is also enhanced by the fact that these are biodegradable
and emit low GHG as oppose to petroleum-based products. Other biofuels are
produced from biomass; however, PHAs could be produced from agricultural
waste and sewage sludge, thus providing a new insight into the ‘food versus fuel’
controversy. Further work could be carried out to make PHA biofuels more effi-
cient and cost effective.
96 Chapter 3
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Figure 3.8 Comparison of GHG emissions and fossil energy requirements of repre-
sentative petroleum and biobased polymers based on 1 kg of resin pro-
duced. Symbols: polystyrene (PS), low-density polyethylene (LDPE),
polyethylene terephthalate (PET), polypropylene (PP), polylactide (PLA),
polyhydroxyalkanoates based on glucose (PHA-G), PHA based on oil
(PHA-O), and PHA based on black syrup (PHA-BS).107
Table 3.4. One area of application where the high cost of PHA can be tolerated in
lieu of its amenable properties is in medical applications. However, if PHAs have to
play a dominant role in the bioplastics market and act as a serious contender for
View Online
Acknowledgement
The author R. Rai greatly acknowledges the financial support from the Quintin
Hogg Foundation of the University of Westminster.
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phase in thermoplastic matrices.9–11 Its low density, highly reduced wear of the
processing machinery, and a relatively reactive surface may be mentioned as
attractive properties, together with their abundance and low price. Moreover,
the recycling by combustion of cellulose-filled composites is easier in compar-
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ison with inorganic filler systems. Nevertheless, such fibres are used only to a
limited extent in industrial practice, which may be explained by difficulties in
achieving acceptable dispersion levels.
From the molecular structure of cellulose given in Figure 4.1, it can be seen
that cellulose is a homopolymer of D-anhydroglucopyranose monomeric units
connected through b (1-4) glycosidic linkages. In general, cellulose can be seen
as a long chain polymer with D-glucose, a sugar, as its repeating units. Since the
glucose units are six-membered rings within a cellulose chain, they are known
as pyranoses. These pyranose rings are joined by single oxygen atoms, acetal
linkages, between the C-1 of one pyranose ring and the C-4 of the next ring. The
glucose units in cellulose polymer are referred to as anhydroglucose units.
Often, in nature, cellulose is associated and mixed with other substances such as
lignin, pectins, hemicelluloses, fats and proteins. Cellulose that is produced by
plants is referred to as native cellulose, which is found in two crystalline forms,
cellulose I and cellulose II.12 Cellulose II, generally occurring in marine algae, is
a crystalline form that is formed when cellulose I is treated with aqueous
sodium hydroxide.13–15 Among the four different crystalline polymorphs, cel-
lulose I, II, III and IV, cellulose I is thermodynamically less stable while cel-
lulose II is the most stable structure.
Liquid ammonia treatment of cellulose I and cellulose II gives crystalline
cellulose III form16–18 and the heating of cellulose III generates the cellulose IV
crystalline form.18 Recently a non-crystalline form known as nematic ordered
cellulose has been described.19 The hydroxyl groups on the cellulose chain that
are in the equatorial position protrude laterally along the extended molecule.
Within the crystalline regions, the extensive and strong inter-chain hydrogen
bonds give the resultant fibres good strength and insolubility in most solvents.
Cellulose has no taste, is odourless, hydrophilic, chiral and biodegradable. It is
tensile strength. This strength is important in cell walls, where the microfibrils are
meshed into a carbohydrate matrix, conferring rigidity to plant cells. It has
interesting sound insulation properties and low thermal conductivity.
106 Chapter 4
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108 Chapter 4
beans), all characterized by double helices: almost perfect left-handed, six-fold
structures, as elucidated by X-ray.
Starch molecules arrange themselves in plants in semi-crystalline granules.
Each plant species has a unique starch granular size: rice starch is relatively
small (about 2 mm) while potato starches have larger granules (up to 100 mm).
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00102
Although in absolute mass only about one quarter of the starch granules in
plants consist of amylose, there are about 150 times more amylose molecules
than amylopectin molecules. Amylose is a much smaller molecule than amy-
lopectin. A typical feature of starch is that it becomes soluble in water when
heated. The granules swell and burst, the semi-crystalline structure is lost and
the smaller amylose molecules start leaching out of the granules. This process is
called starch gelatinization. During cooking, starch becomes a paste and
becomes viscous. During cooling or prolonged storage of the paste, the semi-
crystalline structure partially recovers and starch paste thickens. This is mainly
caused by the retrogradation of the amylose. This process is also responsible for
staling, hardening of bread and water layer on top of a starch gel. Some cul-
tivated plant varieties have pure amylopectin starch without amylose, known as
waxy starch. Waxy starch has less retrogradation; the viscosity of the paste will
be more stable. Also high amylose starch, amylomaize, is cultivated for the use
of its gel strength. The amorphous and partially crystalline rings start from the
centre, called the hilum, and follow each other alternatively. The amorphous
rings are amylose, while the partially crystalline ones are amylopectin. The
macromolecules are oriented in the radial direction. The amorphous region
of partially crystalline rings are formed by those parts of the amylopectin
macromolecule where the chain branches. The crystalline part consists of the
oriented, double helix molecular chain parts of amylopectin. The structure of
amylose and amylopectin are given in Figures 4.3 and 4.4, respectively.
Starch has received considerable attention during the past two decades as a
biodegradable thermoplastic polymer and as biodegradable particulate filler.
Indeed, products from agricultural sources such as starch, offers an attractive
and cheap alternative in developing degradable materials. Starch is not truly
thermoplastic as most synthetic polymers. However, it can be melted and made
to flow at high temperatures under pressure. If the mechanical shears become
too high, then starch will degrade to form products with low molecular weight.
Addition of water or other plasticizers enables the starch to flow under milder
conditions and reduces the degradation considerably. However, the thermo-
chemical stability is strongly due to the addition of plasticizers.50 By itself,
starch is a poor choice as a replacement for any plastic. It is mostly water
insoluble, difficult to process and brittle. In principle some of the properties of
starch can be significantly improved by blending it with synthetic polymers.
Physical incorporation of granular starch or starch derivatives as a functional
additive and filler into synthetic polymers during processing has been largely
used, since the first announcement of using starch in combination with syn-
thetic polymer either as starch gel blends with ethylene acrylic acid copolymer
by Westhoff et al.51 or as a particulate starch dispersion in polyolefin by Griffin
and Priority.52
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110 Chapter 4
4.2.4 Soy Protein Particles
Soy protein isolate (SPI), the major component of soybeans, is readily available
from renewable resources and agricultural processing by-products. Soy pro-
teins are of two types: soy protein concentrates (SPC) (70% protein minimum
dry weight basis) and soy protein isolates (SPI) (90% protein minimum dry
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112 Chapter 4
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aliphatic polyester derived from renewable resources, such as corn starch (in the
USA) or sugarcanes (the rest of the world). Although PLA has been known for
more than a century, it has only been of commercial interest in recent years, in
light of its biodegradability. PLA is a sustainable alternative to petrochemical-
derived products, since the lactides from which it is ultimately produced can be
derived from the fermentation of agricultural by-products such as corn starch
or other carbohydrate-rich substances like maize, sugar or wheat.
Poly(lactic acid) is a versatile polymer made from renewable agricultural raw
materials, which are fermented into lactic acid.1,57 Due to its good bio-
compatibility, biodegradability, mechanical properties and light weight, PLA
has been widely used in many aspects, such as medical applications2,3,58,59 and
automotive parts.4,60 The commercial market for PLA has increased sub-
stantially in recent years. PLA offers advantages of relatively high strength and
ability to be processed in most equipment, but reinforcement is usually needed
for practical applications due to its brittleness.5,6,62 One way to improve the
mechanical and thermal properties of PLA is the addition of fibres or filler
materials.5–7,61–63 Traditional fibres (e.g. glass fibre and recycled newspaper
fibre) and natural fibres (e.g. bamboo and silk fibres) have been used as rein-
forcements to enhance the mechanical properties of PLA.8–10,64–66 PLA is
currently used in a number of biomedical applications, such as sutures, stents,
dialysis media and drug delivery devices. It is also being evaluated as a material
for tissue engineering. Because it is biodegradable, it can also be employed in
the preparation of bioplastic, useful for producing loose-fill packaging, com-
post bags, food packaging and disposable tableware. In the form of fibres and
non-woven textiles, PLA also has many potential uses, for example as
upholstery, disposable garments, awnings, feminine hygiene products and
nappies (diapers). PLA has been used as the hydrophobic block of amphiphilic
synthetic block copolymers used to form the vesicle membrane of
polymersomes.
The basic monomer of PLA is lactic acid, which is derived from starch by
fermentation. Lactic acid is then polymerized to poly(lactic acid), either by
gradual polycondensation or by ring-opening polymerization.67–69 PLA is and
has been frequently used for biodegradable packing materials.70,71 However,
numerous tests have shown that PLA is also suitable as a matrix for the
embedding of fibres in composites. Some products of natural fibre-reinforced
PLA are already established at the market: Jakob Winter (Satzung, Germany)
produces biodegradable urns from flax and PLA by compression moulding.72
NEC Corporation and UNITIKA LTD have announced joint development of
bioplastic composites for mobile phone shells consisting of PLA and 15–20%
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114 Chapter 4
products that can provide PLA with flame retardancy sometimes trigger
problems such as loss of mechanical and thermal properties or degradation of the
polyester matrix, aspects that need to be considered when targeting a potential
application.
Polylactide has still not found any meaningful market acceptance as an
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engineering resin, because of its non-satisfying impact resistance and low heat
distortion temperature. Therefore, manufacturing of PLA lightweight parts
with a high impact resistance would lead to new application fields, e.g. auto-
motive or electrical industry. PLA already has found applications in textile and
medical fields and also in the packaging industry. Besides, PLA is in principle
compostable under certain conditions, that is, in the presence of the right
triggers the material can be degraded into harmless natural compounds.
Nonetheless, the inherent brittleness of PLA has been the main obstacle to
expand its commercial use. Many approaches like plasticization, block co-
polymerization, blending with tougher polymers or rubber have been proposed
but all led to significant decreases in modulus and strength of the toughened
PLA.86
116 Chapter 4
FE-SEM showed that PLA and PVOH formed two immiscible phases with a
continuous PLA phase and a discontinuous PVOH phase. The thermal stability
of the nanocomposites was not improved compared to its unreinforced coun-
terpart, probably because the majority of the whiskers were located in the
PVOH phase and only a negligible amount was located in the PLA phase. The
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118 Chapter 4
play an important role in reinforcing the composites without interfering with
their biodegradability. The SPI/chitin whisker nanocomposites at 43% relative
humidity increased in both tensile strength and Young’s modulus from
3.3 MPa for the SPI sheet to 8.4 MPa and from 26 MPa for the SPI sheet to
158 MPa.
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120 Chapter 4
119
Cheng and co-workers have prepared chicken feather fibre (CFF)/rein-
forced poly(lactic acid) (PLA) composites using a twinscrew extruder and an
injection moulder. The tensile moduli of CFF/PLA composites with different
CFF content (2, 5, 8 and 10 wt%) were found to be higher than that of pure
PLA, and a maximum value of 4.2 GPa 16% was attained with 5 wt% of CFF
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00102
4.4 Applications
All green nanocomposites are low-cost materials. These low-cost green com-
posites were found to have mechanical strength and properties suitable for
applications in housing construction materials, furniture and automotive parts.
The influence of fibre treatment on the properties of biocomposites derived
from grass fibre. Studies carried out in the past decades have demonstrated that
green composites materials should combine high mechanical and other essential
operational and technological properties (e.g. stability, low gas permeability,
environmental safety, easy moulding) with biodegradability.. Recent work on
biocomposites reveals that in the most cases the specific mechanical properties
of biocomposites are comparable to widely used glass fibre reinforced plastics.
In order to be competitive, ecofriendly composites must have the same desir-
able properties as obtained in conventional plastics. The most important fac-
tors to the formation of a successful green composites material industry include
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122 Chapter 4
cost reduction as well as public and political acceptance. Existing green and
environmental friendly composites materials are mainly blended with different
materials with an aim to reduce cost and to tailor the product for some specific
applications.
Application of green composites in natural fibre-reinforced composites will
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00102
broaden their uses. The demand for green and renewable materials continues to
rise. Polymeric composites from renewable resources have occupied major
applications in green packaging. Although these are emerging as alternatives to
existing petroleum derived plastics; the present low level production and high
costs restrict their wide spread applications. The barrier properties of such
degradable polymers can be improved through nano reinforcements. The
incorporation of nanoparticles in a polymer matrix reduces the permeability of
penetrant molecules and thus develops high barrier composites. Nanocompo-
sites especially green nanocomposites or nanocomposites obtained from
renewable resources are an emerging new class of materials, total environ-
mental/economical impacts of which need to be studied to prove the industrial
and environmental potential of targeted nanocomposites for automotive
applications. Biopolymers combined with suitably organically modified clays
will result in environmental friendly nanocomposites. The need of green and
biodegradable plastics has been increased during the past decades not only due
to the increasing environmental concerns but also for its biomedical applica-
tions. It is now well evident that polymer / plastics waste management through
biodegradation or bio-conversion is the most suitable solution for ‘Plastic
Waste Management’ among the other traditional methods like incineration,
pyrolysis and landfilling. Several means have been used to achieve the biode-
gradability/biodeteriobility /Photo-biodegradability in polymers.
4.5 Conclusion
As a result of environmental awareness and the international demand for green
technology, green composites have the potential to replace present petro-
chemical-based materials. They represent an important element of future waste
disposal strategies. In true green nanocomposites, both the reinforcing material
(such as a natural fibre) and the matrix are biodegradable. Cellulose, chitin and
starch are the most abundant organic compounds in nature; they are also green,
inexpensive, biodegradable and renewable. They obviously receive a great
attention for non-food applications. The use of natural fibres instead of tra-
ditional reinforcement materials, such as glass fibres, carbon and talc, provides
several advantages including low density, low cost, good specific mechanical
properties, reduced tool wear and biodegradability. Important applications
include packaging, wide-ranging uses from environmentally friendly biode-
gradable composites to biomedical composites for drug/gene delivery, tissue
engineering applications and cosmetic orthodontics. They often mimic the
structures of the living materials involved in the process in addition to the
strengthening properties of the matrix that was used but still providing green
View Online
coir, etc. Green fibres can be combined with traditional resins or newer plant-
based resins. The result is a plant-based alternative for many traditional steel
and fibreglass applications. Advantages of green nanocomposites over tradi-
tional composites are reduced weight, increased flexibility, greater mould-
ability, reduced cost, better sound insulation and their renewable nature.
References
1. R. L. Crawford, Lignin Biodegradation and Transformation. Wiley, New
York (1981). ISBN 0-471-05743-6.
2. R. Young, Cellulose Structure Modification and Hydrolysis. Wiley, New
York (1986). ISBN 0471827614.
3. D. Klemm, H. Brigitte, F. Hans-Peter and B. Andreas, J. Chem.-Inform.,
2005, 36, 36.
4. D. M. Updegraff, J. Anal. Biochem., 1969, 32, 420.
5. Cellulose Encyclopedia Britannica, Encyclopedia Britannica Online,
Retrieved January 11, 2008 (2008).
6. A. K. Bledzki, S. Reihmane and J. Gassan, J. Appl. Polym. Sci., 1996, 59,
1329.
7. K. G. Satyanarayana, K. Sukumaran, P. S. Mukherjee and C. P. Pavi-
tharan, J. Compos., 1990, 12, 117.
8. A. Bismarck, S. Mishra and T. Lampke, Plant Fibres as Reinforcement for
Green Composites, CRC Press, Boca Raton, FL, (2005), pp. 37–108.
9. C. Klason, J. Kubat and H. E. Stromvall, J. Polym. Mater., 1985, 11, 9.
10. P. Zadorecki and A. J Michell, J. Polym. Compos, 1989, 10, 69.
11. D. Maldas, B. V. Kokta, R. Raj and G. C. Daneault, J. Polym., 1988, 29,
1255.
12. S. Kuga and R. M. Brown, J. Carbohydr. Res., 1988, 180, 345.
13. K. R. Z. Andress, J. Phys. Chem. Abt. B, 1929, 4, 190.
14. J. Blackwell and F. Kolpak, J. Macromol., 1976, 9, 273.
15. H. Chanzy, Y. Nishiyama and P. Langan, J. Am. Chem. Soc., 1999, 121,
9940.
16. S. Watanabe, J. Ohkita, J. Hayashi and A. Sufoka, J. Polym. Lett, 1975,
13, 23.
17. A. J. Sarko, J. Southwick and J. Hayashi, J. Macromol., 1976, 9, 857.
18. H. Chanzy and A. Buleon, J. Polym. Sci. Polym. Phys. Ed, 1980, 18, 1209.
19. E. Togawa, R. M. Brown and T. J. Kondo, J. Biomacromol., 2001, 2,
1324.
20. J. Li, J. F. Revol and R. H. Marchessault, J. Appl. Polym. Sci., 1997, 65,
373.
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21. Y. Yamaguchi, T. T. Nge, A. Takemura, N. Hori and H. Ono, J. Bio-
macromol., 2005, 6, 1941.
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23. N. L. Yusof, A. Wee, L. Y. Lim and E. Khor, J. Biomed. Mater. Res. A,
2003, 66, 224.
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76. D. Garlotta, J. Polym. Environ., 2001, 9, 63.
77. R. E. Drumright, P. R. Gruber and D. E. Henton, J. Adv. Mater., 2000,
12, 1841.
78. L. Cabedo, J. L. Feijoo, M. P. Villanueva, J. M. Lagaro and E. Gimenez,
Macromol. Symp., 2006, 233, 191.
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128. T. Nishino, K. Hirao, M. Kotera, K. Nakamae and H. Inagaki,
J. Comput. Sci. Technol., 2003, 63, 1281.
129. A. K. Bledzki, A. Jaszkiewicz and V. E. Sperber, Abaca and cellulose fibre
reinforced polypropylene, In: Proceedings of Fourth International Work-
shop on Green Composites, Tokyo, September 2006, pp. 1–5.
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00102
Biopolymer-based Nanocomposites
KIKKU FUKUSHIMA,1 DANIELA TABUANI2 AND
CRISTINA ABBATE3
1
Politecnico di Torino sede di Alessandria, Viale Teresa Michel 5, 15121
Alessandria, Italy; 2 Consorzio PROPLAST, Strada Savonesa 9, 15057
Rivalta Scrivia, Alessandria, Italy; 3 Dipartimento di Scienze Agronomiche,
Agrochimiche e delle Produzioni animali (DACPA), University of Catania,
Via S. Sofia 98, 95123 Catania, Italy
5.1 Introduction
Synthetic polymers are used for various purposes, especially in the packaging
industrial sector; however, the majority of these materials constitutes at present a
serious problem for waste management because more than 80% of plastic pro-
duction is based on polyolefins which are mostly produced from fossil fuels and
after consumption can be discarded into the environment, ending up as practi-
cally eternal undegradable wastes.1 In order to solve this problem, biodegradable
polymers have attracted a special attention as the plastics of the 21st century,
since they can be biologically degraded and, therefore, can be considered as
environmental friendly materials.2 Among all the studied biodegradable poly-
mers, polyesters play a key role due to the presence of highly hydrolysable ester
bonds in their chemical structure,3 making these polymers highly subject to
degradation in humid environments as well as in natural conditions considering
that enzymes able to degrade esters (esterases) are ubiquitous in nature.
Among these biodegradable polyesters, poly(lactic acid) (PLA) and
poly(e-caprolactone) (PCL) show a great potential for different applications,
such as in agriculture and in everyday life as biodegradable packaging material,4
129
130 Chapter 5
because of their facile availability, good biodegradability and good mechanical
properties. PLA (from L-lactic acid, D-lactic acid or mixtures of both) can be
obtained by means of a fermentation process using glucose from corn. The
proportion of the L- and D- isomeric forms will determine the properties of
the polymer, i.e. if the material is amorphous or semi-crystalline.5,6 Conversely,
PCL is a linear and partially crystalline polymer, synthesized by ring-opening
polymerization of cyclic e-caprolactone in presence of a catalyst.1
As far as polymer hydrolytic degradation is concerned, most of the studies on
PLA have concentrated on abiotic hydrolysis, which has been reported to take
place mainly in the bulk of the material rather than its surface.7–10 This process
has been described as an autocatalytic hydrolysis of PLA occurring homo-
geneously along sample cross-section: the formation of polylactic acid oligo-
mers, which follows the chain scission, increases the carboxylic acid end groups
concentration in the degradation medium, thus rendering the hydrolytic
degradation of PLA a self-catalysed and self-maintaining process.8,9,11
Concerning PLA biodegradability, it has been shown that PLA is naturally
degraded in soil or compost,12–14 as the resulting products of its hydrolytic
degradation in these media can be totally assimilated by microorganisms such
as fungi or bacteria.2,15–17 Even more, the presence of compost microorganisms
in the degradation medium seems to accelerate the hydrolytic degradation of
PLA compared to the degradation in the corresponding sterile medium.18
In the case of PCL, it has been reported that this is a relatively stable material
against abiotic hydrolysis;19 however, it has been found that this polymer can be
easily degraded by microorganisms widely distributed in different environments
such as compost and soil;19,20 indeed several authors have reported on highly active
bacteria and some fungal phytopathogens towards PCL degradation.19,21–25
Contrary to PLA, it has been shown that the biodegradation of PCL proceeds by a
rapid weight loss through surface erosion with a minor reduction of molecular
weight in contrast to its abiotic hydrolysis, which proceeds by a strong reduction in
molecular weight combined with minor weight losses.19
The main limitations of these biodegradable polymers towards their wider
application are their relatively low thermal and mechanical resistance and
limited gas barrier properties, which limit their access to certain industrial
sectors, such as food packaging, in which their use would be justified when
biodegradability is required.26 Nevertheless, the above drawbacks could be
overcome by enhancing their properties through the use of filler and/or addi-
tives. In the last two decades, the addition of nanofillers to polymers has
attracted great attention for the potentiality of these materials to improve a
high number of polymer properties; for example, polymer layered silicate
nanocomposites, because of the nanometer size of the silicate sheets, exhibit,
even at low filler content (1–5 wt%), markedly improved mechanical, thermal,
barrier and flame retardant properties, in comparison to the unfilled matrix and
to the more conventional microcomposites.3,27–29
Several authors have recently reported on the preparation and character-
ization of biodegradable polymer-based nanocomposites. Paul et al.7 reported
the preparation of PLA/montmorillonite systems by melt intercalation using a
Biopolymer-based Nanocomposites 131
montmorillonite modified with bis-(2-hydroxyethyl) methyl (hydrogenated
tallow alkyl) ammonium cations. Chang et al.29 reported the preparation of
PLA-based nanocomposites with three different kinds of layered silicates, via
solution intercalation method in N-dimethylacetamide, obtaining intercalated
nanocomposites independently of the clay nature. Pantoustier et al.30 used the
in situ intercalative polymerization method to obtain PCL-based nanocompo-
sites with high dispersion level. Di et al.31 reported the preparation of PCL/
layered silicate nanocomposites using a twin-screw extruder, determining a
dependence of clay dispersion on the processing conditions.
Concerning the biodegradation of polymer nanocomposites, recently most of
researchers report that nanofillers are able to accelerate polymer biodegradation. In
particular, Ray et al.32 conducted a respirometric test to study the degradation of
PLA and its organically modified layered silicate nanocomposites in a compost
environment, observing a catalytic effect of some of these clays towards PLA bio-
degradation, whereas other nanocomposites presented the same degradation level
of neat PLA. They concluded that the observed differences can be related to the
different types of clays used which could influence the hydrolysis of the esters bonds.
In other studies, Lee et al.33 and Paul et al.34 have found an increased PLA
biodegradation rate in compost due to the presence of nanoparticles. This
behaviour was attributed to the high relative hydrophilicity of the clays,
allowing an easier permeability of water into the bulk material, thus possibly
accelerating the hydrolytic degradation process of the polymer matrix.
Biodegradation of PCL-based nanocomposites has been scarcely studied until
now. Tetto et al.35 reported that organically modified layered silicates accelerate
PCL biodegradation. Contrarily, Maiti et al.36 reported that the biodegradation of
PCL-based nanocomposites can be depressed by the presence of nanoparticles,
probably due to an improvement of the barrier properties of the polymer matrix.
In summary, despite the addition of nano-sized particles that would poten-
tially confer multifunctional enabling properties to these biodegradable poly-
mers for several industrial applications, until now very few works have dealt
with the investigation of the real impact of nanoparticles on biopolymer
degradation rate and mechanism.
On this background, the general aim of this work is to study the addition
of two different nanoparticles on mechanical properties and biodegradation
trends of poly(lactic acid) (PLA) and poly(e-caprolactone) (PCL), playing
particular attention on the influence of polymer filler affinity.
5.2 Experimental
5.2.1 Materials and Methods
Poly(lactic acid) (PLA, 4042D) and poly(e-caprolactone) (PCL, CAPAs6500)
of commercial grade were supplied by NatureWorks and SOLVAY S.A.
respectively. Commercial modified montmorillonites were supplied by South-
ern Clay (USA) (CLOISITE 30B) and Süd-Chemie (Germany) (NANOFIL
804). The characteristics of the clays are listed in Table 5.1.
132 Chapter 5
37
Table 5.1 Characteristics of clays.
Commercial name Modifier structure Code
NANOFIL 804 HT O H
NAN804 (nB1)
+ n
N
R O H
n
Prior to the mixing step, PLA and PCL were dried at 50 1C under vacuum
for 8 h and the clays at 90 1C under vacuum for 6 h. Nanocomposites
were obtained at 5% clay loading by melt blending using an internal mixer
(Rheomix-Brabender OHG 47055) with a mixing time of 5 min at 75 1C and
165 1C for PCL and PLA respectively. The mixing was performed at two
different rotor speeds: 30 rpm in the loading step and 60 rpm during mixing.
The batch was extracted from the mixing chamber manually, allowed to cool to
room temperature in air and ground in a rotatory mill. Sheets were obtained by
compression moulding in a hot-plate hydraulic press at 120 1C (PCL) or at
210 1C (PLA) and allowed to cool to room temperature under pressure. All
characterizations were made on 0.6 mm films, except the WAXS analysis
which was performed on 3 mm films. Biodegradation tests were performed on
0.13 mm films.
The characterization of the materials was performed in order to evaluate
their morphology (X-ray diffraction and electron microscopy) and mechanical
properties through dynamic mechanical analysis.
Wide angle X-ray spectra (WAXS) were recorded using a Thermo ARL
diffractometer X-tra 48, at room temperature, in the range 1–301 (2y) (step
size=0.021, scanning rate ¼ 2 s per step) by using filtered Cu Ka radiation
(l ¼ 1.54 Å).
Scanning electron microscopy (SEM) was carried out on the cryogenic
fracture surfaces of the specimens coated by sputtering with gold, using a Leo
14050 VP SEM apparatus equipped with energy dispersive spectroscopy (EDS).
Transmission electron microscopy (TEM) was performed using a high-
resolution transmission electron microscope (JEOL 2010). Ultrathin sections
about 100 nm thick were cut with a Power TEOMEX microtome equipped with
a diamond knife and placed on a 200-mesh copper grid.
Dynamic mechanical thermal analysis (DMTA) was performed on com-
pression moulded 6 20 0.6 mm3 films, using a DMTA TA Q800 in tension
film clamp. The analysis temperature range was from +25 to +90 1C (in the
case of PLA) and from –150 to +50 1C (in the case of PCL), at a heating rate of
Biopolymer-based Nanocomposites 133
1
2 1C min , 1 Hz frequency, preload of 0.4 N (for PCL) and 0.01 N (for PLA),
in strain controlled mode and 15 micron of amplitude. All tests were carried out
according to the International Standard UNI EN ISO 6721.
(a)
d = 4.1 nm
PLA+ 5%CLO30B
Intensity (a.u.)
d = 3.3 nm
PCL+ 5%CLO30B
d = 1.8 nm
CLO30B
500 a.u.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
2θ(°)
(b)
d = 4.7 nm
Intensity (a.u.)
d = 1.8 nm PCL+5%NAN804
d = 4.7 nm
d = 1.8 nm
NAN804
150 a.u.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
2θ(°)
Figure 5.1 WAXS patterns of CLO30B, NAN804 and nanocomposites of PLA and
PCL with (a) 5% CLO30B and (b) 5% NAN804.38 Reproduced from
K. Fukushima et al. by permission of Elsevier Science Ltd., UK.
(a)
(b)
(a)
(b)
(a)
(b)
Figure 5.4 TEM patterns of (a) PLA + 5% CLO30B and (b) PLA + 5% NAN804.38
Reproduced from K. Fukushima et al. by permission of Elsevier Science
Ltd., UK.
Associating these observations with WAXS and SEM results, we can conclude
that the significant decrease of the peak intensity of NAN804 in the composite
spectra should be due to disordering by intercalation rather than by exfoliation
of the silicate layers.
Evaluating WAXS, SEM and TEM results, it is possible to say that the
dispersion and affinity of CLO30B in both polymers is higher than what
obtained with NAN804 and this is attributed to higher interactions between
the polymers and CLO30B, originated from the hydrogen bonding between
the carbonyl groups of the polymers and the hydroxyl groups of CLO30B
organic modifier.1,33,38 Although both CLO30B and NAN804 are character-
ized by the presence of hydroxyl groups in the organic modifier, it can be
assumed that the shorter alkyl chains of CLO30B organic modifier make
138 Chapter 5
hydroxyl groups more available for interactions with the polymers than the
olygomeric structure of NAN804, thus allowing for a higher dispersion
degree.38
Figure 5.5 PLA and nanocomposites based on CLO30B (+CLO30B) and NAN804
(+NAN804) before degradation (0 weeks) and after 3 and 4 weeks of
degradation in compost at 40 1C.37 Reproduced from K. Fukushima et al.
by permission of Elsevier Science Ltd., UK.
Figure 5.6 PCL and nanocomposites based on CLO30B (+5% CLO30B) and
NAN804 (+5% NAN804) before degradation (0 weeks) and after 3 and 4
weeks of degradation in compost.42 Reproduced from K. Fukushima et al.
by permission of Elsevier Science Ltd., UK.
Biopolymer-based Nanocomposites 141
the compost along all the specimen surface and/or to an inhomogeneous
growth of the microorganisms on the sample surface, leading to fragmentation
of the specimen as well as loss of degradation products by migration into the
compost.42
The significant and fast degradation, previously observed by visual obser-
vation and through optical micrographs, can be confirmed by the important
weight decreases obtained by all PCL-based materials in short times in compost
(Figure 5.8), observing an almost complete specimen weight loss after 8 weeks
and indicating the considerable fragmentation level of PCL matrix in this
medium.42 This trend is not influenced by the presence of the clays.
It is worth noticing the different rate and mechanism of degradation
observed for PLA- and PCL-based materials in the same compost. These dif-
ferences could be associated to the different polymer chemical structure of these
Figure 5.7 Optical micrographs of (a) PCL, (b) PCL+CLO30B and (c) PCL+NAN804
after 6 weeks in compost.42 Reproduced from K. Fukushima et al. by
permission of Elsevier Science Ltd., UK.
100
90
80
Residual mass (%)
70
60
50
40
30
PCL
20
PCL + 5%CLO30B
10 PCL + 5% NAN804
0
0 1 2 3 4 5 6 7 8
Time (weeks)
Table 5.4 SEC data on PLA and its nanocomposites at different times of
degradation in compost. Data extracted from K. Fukushima
et al.37 by permission of Elsevier Science Ltd., UK.
Week 0 Week 4 Week 17
Mn Mw Mn Mw Mn Mw
Sample (g/mol) (g/mol) (g/mol) (g/mol) (g/mol) (g/mol)
PLA 72 743 149 593 67 300 138 181 32 775 113 096
PLA+CLO30B 64 270 135 768 40 412 120 642 13 461 64 817
PLA+NAN804 47 528 103 217 34 559 74 128 27 911 67 341
Biopolymer-based Nanocomposites 143
0 10 20 30 40 50 60 70 80 90 100
Residual Number Average Molecular Weight [%]
Figure 5.9 Residual number average molecular weight of PLA and its nanocompo-
sites before and after 4 and 17 weeks of degradation in compost.
PCL 73 751 115 555 28 989 109 405 21 464 100 288
PCL+CLO30B 72 074 123 179 71 753 123 938 37 737 109 344
PCL+NAN804 77 569 107 656 43 577 115 082 26 034 105 224
0 10 20 30 40 50 60 70 80 90 100
Residual Number Average Molecular Weight [%]
Figure 5.10 Residual number average molecular weight of PCL and its nano-
composites before and after 4 and 6 weeks of degradation in compost.
Mw decreases in unfilled PCL from 115 555 g mol1 to 100 288 g mol1,
losing ca. 13% after 6 weeks (see Table 5.5) and the losses for nanocomposites
were 11% and 2% in the case of CLO30B and NAN804, respectively.
The larger decrease of Mn as compared to Mw indicates that in the degraded
PCL samples, the quantity of polymer chains shortened by hydrolysis reaction
is larger than that of low molecular weight present in the original sample.
This is noticeable by observing that Mw is only slightly lower in the degraded
specimens.42 This behaviour was previously obtained in the case of PLA-based
materials; however, the mechanistic explanation for the higher decreases of Mn
as compared to Mw upon degradation is different in the two situations because,
Biopolymer-based Nanocomposites 145
in the studied time of degradation, weight losses of PLA upon degradation were
insignificant, whereas those of PCL were extensive. Thus, preferential release of
low molecular weight polymer chains into the compost must be taken into
account in PCL.
Interestingly enough and contrarily to what obtained for PLA-based mat-
erials, the higher decreases of Mn and Mw were found for neat PCL as compared
to nanocomposites, indicating that, in this case, the presence of the nano-
particles tends to delay the polymer degradation process. Considering that PCL
degradation seems to mainly proceed from the surface to the interior of the
sample, it is possible that the presence of the silicate layers acts as a barrier
towards microbial attack on PCL ester groups, slowing down the diffusion of
enzymes into the polymer matrix. We can assume that adhesion of PCL and/or of
enzymes macromolecules from compost microorganisms to clay layers could
partially delay the polymer degradation due to less suitable macromolecular
conformations for hydrolysis reactions.42 Indeed, the higher delaying effect is
observed upon the addition of CLO30B and possibly because of its higher
dispersion level in the PCL matrix as compared to NAN804.
In summary, the effect of nanoparticles on polymer biodegradation was
found to be highly dependent on the polymer matrix type and in particular on
the type of degradation mechanism to which the polymer is subjected.
5.4 Conclusions
This work addresses the study of the effect of two different organically modified
montmorillonites (CLO30B, NAN804) on several properties and biodegrada-
tion trends of polylactic acid (PLA) and poly(e-caprolactone) (PCL). As far as
mechanical properties are concerned, the highest increases in storage modulus
were obtained for PLA nanocomposites compared to PCL ones, due to higher
polymer/filler interactions.
For both polymers, the most considerable enhancements were found upon
the addition of CLO30B because of its high dispersion and attributed to the
increased number of organic modifier hydroxyl groups available to promote
polymer/fillers interactions.
The degradation of PLA, PCL and their nanocomposites in compost under
controlled conditions was also studied in this work. According to most litera-
ture, the addition of clays in biopolymers could accelerate their biodegrad-
ability level in compost; however, in this work we could observe that this is not
an iron rule as nanoparticles can have different effects on the polymer degra-
dation trend depending on the degradation mechanism of neat polymer and on
the level of clay dispersion into the polymer.
Two different mechanisms of degradation in compost were assumed for neat
PLA and PCL, predominant bulk degradation for PLA and a preferentially
surface degradation for PCL. The type of dominating degradation mechanism
seems to depend on both the structure of the polymer, physical properties (glass
146 Chapter 5
transition temperature) and on the environment it is subjected to. In summary,
nanoparticles seem to affect polymer degradation as follows:
(1) When the degradation mainly occurs through a bulk mechanism (PLA),
the addition of nanoclays was found to increase the polymer degradation
rate because of the presence of hydroxyl groups belonging to the silicate
layers; this phenomenon can be particularly evident for the highest
dispersed clay in the polymer (PLA/CLO30B).
(2) When the degradation occurs through a mainly surface mechanism
(PCL), highly dispersed layered clays can partially delay the polymer
degradation rate probably due to a more difficult route for micro-
organisms in order to attack the polymer ester groups; this last phe-
nomenon can be more evident at higher clay dispersion levels in the
polymer (PCL/CLO30B).
Acknowledgements
The authors would like to thank gratefully Prof. Giovanni Camino and Prof.
Mara Gennari for their great support and guidance throughout this work. The
authors would like to acknowledge Dr Orietta Monticelli from Genoa
University for TEM characterizations, Dr Paola Rizzarelli and Loredana
Ferreri from CNR ICTP (University of Catania) for SEC characterizations,
Dr Michele Negre from Università degli Studi di Torino for degradation tests
in compost, as well as EU NoE ‘NANOFUN POLY’ for financial support.
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CHAPTER 6
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00149
Biodegradable Polyesters:
Synthesis and Physical
Properties
JASNA DJONLAGIC AND MARIJA S. NIKOLIC
6.1 Introduction
Biodegradable polymers have come into the focus of research and development
as a proposed solution to the increasing environmental problems associated
with the disposal of traditional commodity plastics.1–10 The use of a polymeric
material which will disintegrate in nature after it has served its function, in
contrast to the currently used, long-lasting polymeric materials, would certainly
be the best solution. From the view point of depletion of petrochemical
resources, the production of such biodegradable polymeric material from
renewable sources would positively contribute to the issue of sustainable
material development.11 Additionally, in the field of medicine, there is also a
growing requirement for degradable polymeric materials which could tem-
porarily serve their function, after which they would be eliminated from the
body.12–17 Thus, environmental protection and biomedicine are the two main
fields of development and application of biodegradable polymers.
In order to be biodegradable, polymers have to contain groups in the
polymer main chain which can undergo scission under biological conditions.
Polyesters are a class of polymers which contain hydrolytically labile ester
149
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150 Chapter 6
linkages. Thus, they are susceptible to degradation in biological environments,
where the conditions for hydrolysis are readily met. Polyester hydrolysis can
proceed with the help of enzymes produced by certain organisms; however, they
can also degrade, at least partially, without the catalytic action of enzymes.
Nevertheless, polyesters which predominately undergo abiotic hydrolysis are
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00149
152 Chapter 6
too large to penetrate into the polymer matrix and their action is restricted only
to the surface. Microorganisms which promote polyester degradation can act in
a mechanical, chemical or enzymatic way.23 The polymeric material is first
extracellularly fragmented into substances which can enter the cells of micro-
organisms, where they are finally mineralized. The actions of microorganisms
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154 Chapter 6
together with a high hydrolytic instability, it was the first synthetic polyester
which appeared on the market as a biodegradable suture material. It was
developed as the synthetic absorbable suture, Dexons, in 1962, by the
American Cyanamid Co.25 Fibers made of PGA are very stiff, thus copoly-
merization is often performed to modify the mechanical properties of the
resulting fibers.16 The self-reinforced PGA, which refers to a composite mate-
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00149
Scheme 6.2 Structural formulas of the two isomeric forms of lactic acid.
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can be obtained in high purity. Thus, the monomer for the production of PLA
is available from renewable and low-cost substances, such as wheat, rice bran,
potato and corn starch and other biomasses.32,33 One advantage of the chiral
structure of lactic acid is the possibility of tailoring the final properties of the
synthesized PLA through its stereostructure by simply changing the ratio of the
D- and L-enantiomers in the polymer backbone. The polymerization of either
enantiomer leads to semi-crystalline polymers (PLLA or PDLA), while
amorphous PDLLA is obtained from a racemic mixture of the monomers and
this difference in crystallinity has profound impact on all properties of PLA.
PLA can be obtained in two ways: through direct polycondensation of the
hydroxy acid or by ROP of cyclic lactide monomer. The different reaction
pathways to PLA are depicted in Scheme 6.3. Different nomenclatures of
polymers obtained by the different routes are often observed in the literature:
those obtained from lactic acid by direct polycondensation are referred to as
poly(lactic acid), while those obtained from lactide monomer by ROP are
referred to as poly(lactide). The general abbreviation used in both cases is PLA.
Direct polycondensation in the bulk through the reaction of hydroxy and
carboxy functionalities of the lactic acid monomer with the elimination of water
molecule has all the drawbacks associated with the character of an equilibrium
step-growth polymerization. In addition, employing this polymerization
method, the stereoregularity of the polymer cannot be controlled. This poly-
merization technique involves the use of a catalyst and reduced pressure.34–37
156 Chapter 6
Oligomers obtained in the direct polycondensation reaction have a molecular
weight of a few tens of thousands, although there are reports where higher
molecular weight PLLA of up to 100 000 g mol 1 were obtained by the use of a
binary catalyst based on Sn(II) activated with proton acids.38 The oligomeric
PLA obtained in the direct polycondensation process is of interest in drug
release systems;39,40 however, it is of little practical importance for the majority
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158 Chapter 6
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00149
Scheme 6.4 Mechanisms of the cationic, anionic and coordination insertion ROP
of lactide.
The properties of PLA depend strongly on the ratio and distribution of the
stereoisomers comprising the polyester chain. In addition to the dependence on
stereoregularity, the properties of PLA depend on the molecular weight as well
as on the annealing and processing conditions.60–62 Homopolymers poly(L-
lactide), PLLA and poly(D-lactide), PDLA, are semicrystalline. PLLA
crystallizes in the a-, b- and g-form, of which the a-form is the most common.
PLLA in the a-form has a pseudo-orthorombic unit cell with lattice dimensions
a=1.066 nm, b=0.616 nm and c=2.888 nm.63 By the introduction of other
stereoisomer into the polymer chain, the crystallization is hindered and finally
completely lost, resulting in an amorphous polymer.64–66 Crystallization of
stereocopolymers depends not only on the composition, but also on the
distribution of stereoisomers within polymer chain.67 The melting temperature
of enantiomeric pure PLLA is 170–180 1C and the glass transition tempera-
ture is around 60 1C. The melting temperature decreases with the introduction of
the second enantiomer and can be lowered by as much as 50 1C.65,67 When PLLA
is blended with PDLA, for certain blend compositions, a stereocomplex can be
formed which is accompanied with an increase in the melting temperature of up
to 50 1C compared to the homopolymers. The PLA stereocrystals have a triclinic
unit cell with the lattice dimensions a=b=1.498 nm, c=0.87 nm, a=b=901 and
g=1201.68 The glass transition temperature, besides molecular weight, is also
dependent on the stereostructure of the PLA and is lower for stereoco-
polymers. The solubility of PLA also depends on composition. PLLA is soluble
in chlorinated and fluorinated solvents, dioxane, furan and dioxolane. Stereo-
copolymer, PDLLA is also soluble in acetone, pyridine, ethyl lactate, tetra-
hydrofuran, xylene, ethyl acetate, dimethylsulfoxide, N,N-dimethylformamide
and 2-butanone. Non-solvents for both PLAs are water, alcohols and hydro-
carbons, such as hexane and heptane.
Semicrystalline PLLA in terms of mechanical properties, having a high
tensile modulus of around 3–4 GPa, flexural modulus of 4–5 GPa, tensile
strength of 50–70 MPa and low elongation at break of around 4%, is a stiff,
brittle and hard polymer.69–71 However, depending on the degree of crystal-
linity, molecular weight and stereoregularity, the mechanical properties can
vary so that elastic and soft PLAs can be obtained.60 The tensile strength of
PLLA increased from 55 to 59 MPa with increasing molecular weight from
23 000 to 67 000 g mol 1, whereas PDLLA stereocopolymers of molecular
weight from 47 500 to 114 000 g mol 1 had lower tensile strengths in the range
from 43–53 MPa. With the increase in crystallinity achieved by annealing, the
mechanical properties of PLLA were further increased, especially the flexular
and tensile modulus.60 Due to its good mechanical properties, PLLA is a good
candidate as a biomaterial for load bearing applications and for articles where
rigidity and strength are required.12 PLA, with its high modulus and low
elongation at break has very similar mechanical properties to poly(styrene),
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160 Chapter 6
72
which is a strong and relatively brittle material. The mechanical properties of
PLA recommend this polymer as a good replacement for non-degradable
commodity plastics in applications such as packaging. However, in order to be
used as a soft film, the addition of plasticizers to reduce Tg is necessary, and a
number of plasticizers, preferably biodegradable, have been investigated in
order to modify the properties of PLA.69,73,74
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00149
6.3 Poly(e-caprolactone)
6.3.1 Synthesis of Poly(e-caprolactone)
Poly(e-caprolactone), PCL, comprised of hexanoate repeating units, is the most
important synthetic biodegradable aliphatic polyester among the poly(o-
hydroxyalkanoate)s (Table 6.1). It can be synthesized by polycondensation of
the corresponding hydoxy acid: 6-hydoxycaproic (6-hydroxyhexanoic) acid.
However, this method of synthesis has been less investigated for the production
of PCL because of all the shortcomings inherent to the direct polycondensation
reaction. Thus, poly(e-caprolactone) is mainly produced by ROP of the cyclic
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162 Chapter 6
monomer e-caprolactone. This monomer is manufactured by the oxidation of
cyclohexanone with peracetic acid (Scheme 6.5).
Depending on the employed initiator/catalyst system, the ROP of e-capro-
lactone can proceed via anionic, cationic or coordination–insertion mechanisms
(Scheme 6.6). The reaction can be performed in bulk, solution or as an emul-
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00149
Scheme 6.6 Mechanisms of the initiation step for cationic, anionic and coordination-
insertion ROP of e-caprolactone.
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164 Chapter 6
110
this procedure. Sn(Oct)2 is active at higher temperatures which promotes
intermolecular and intramolecular transesterification reactions and broadens
the molecular weight distribution.105
PCL can also be obtained by free radical polymerization of 2-methylene-
1,3-dioxepane initiated with AIBN.111,112 However, this reaction pathway
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166 Chapter 6
degradation. In the second stage of PCL degradation, weight loss occurs,
accompanied with a deceleration of the molecular weight decrease (rate of
chain scission) due to the increased crystallinity of the sample.101 When the
molecular weight of PCL falls to 3000 g mol 1, small pieces of PCL can be
intracellulary degraded in the phagosomes of macrophages, giant cells or
fibroblasts in less than 13 days at some implant sites.156 The sole metabolite
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00149
besides water was 6-hydroxycaproic acid derived from the complete hydrolysis
of the polyester. The product of PCL hydrolysis, 6-hydroxycaproic acid and
further coenzyme A, can enter the citric acid cycle and be eliminated from the
body.104 Some recent studies showed that PCL is excreted from the body
immediately after it is metabolized and does not accumulate in any body
organ.157 Investigations on PCL-based scaffolds showed that the degradation
pathway of these devices, under simulated physiological conditions and in vivo,
follows the general trend of bulk chemical degradation.158,159 Due to its slow
degradation in vivo, compared to PLA and PGA, PCL is more suitable for
long-term implants.160
6.4 Poly(hydroxyalkanoate)s
6.4.1 Synthesis of Poly(hydroxyalkanoate)s
Poly(hydroxyalkanoate), PHA, is a general name for a large family of poly-
esters which are produced directly by microorganisms from renewable resources.
Being produced by microorganisms, they are inherently biodegradable. The
first such polymer to be discovered by Lemoigne in the 1920s, produced by
bacteria Bacillus megaterium, was poly(3-hydroxybutyrate), P(3HB), which is
the PHA most often found in nature.161 In 1974, with the discovery of the
copolymer poly(3-hydroxybutyrate)-co-poly(3-hydroxyvalerate), P(3HB-co-
3HV), it was recognized that other 3-hydroxyalkanoate units can be incorpo-
rated in the polyester chain.162 By varying the type of microorganisms and
carbon source, different monomers can participate in copolymer building.
P(3HB) and other PHAs are stored in the microbe cells and serve as an
intracellular energy and carbon storage material, similar to the starch and
glycogen in other living systems. The formation of polymer is usually induced
by the lack of essential nutrients such as sulfate, ammonium, phosphate,
potassium, iron, magnesium or oxygen and in the presence of a source of excess
carbon, such as sugars. Furthermore, some species which accumulate PHAs
during growth, without nutrient limitations, have been found. The polymer
accumulates in the cytoplasm of the cells in the form of discrete granules, the
number and size of which can vary depending on the species. The size of the
granules range from 0.2 to 0.5 mm and the polymer inside is in the amorphous
state, which is easily transformed to crystalline form upon any physical
treatment.163,164
The general formula of poly(hydroxyalkanoate)s is given in Scheme 6.8. The
majority of PHAs have the same three-carbon backbone but differ in the length
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of the alkyl groups attached at the b position. PHAs are divided into two
classes: short chain length PHA (sclPHA, having from 3 to 5 carbon atoms in
the monomeric unit) and medium chain length PHA (mclPHA, having from 6
to 14 carbon atoms in the monomer unit). Polymers with 4-hydroxybutyric acid
moieties have also been prepared and are considered as good candidates for
medical applications.165,166 Some other PHAs, the structures of which are
derived from 5-hydroxy and 6-hydroxy acid repeating units, have also been
isolated. More than 100 monomers have been found to participate in the
construction of different PHAs.167,168
Depending on the size and type of the pendant alkyl group and the com-
position of the polymer, the properties of PHAs can vary over a broad range,
from rigid plastics to tough elastomers.169 The versatility of PHAs can be
increased by different types of chemical modifications due to possibility of
introducing different functionalities, such as unsaturated groups as well as
bromo-, hydroxyl-, methyl-branched and aromatic units, into the chemical
structure of PHA by altering the type of carbon source and microorganisms
used for their production.170 For example, a biodegradable rubber of microbial
origin was prepared by cross-linking of an unsaturated polyester.171
The molecular weights of PHAs range from 2 104 to 3 106 g mol 1
depending on the microorganisms type, growth conditions and type of carbon
source.163 By using a recombinant Escherichia coli under special conditions,
ultra-high molecular weight P(3HB) with molecular weight 4 2 107 g mol 1
has been obtained.172
The biosynthetic production of PHAs is the most important and investi-
gated, and is the only method that has been developed to commercialization.
There are three possible metabolic pathways involved in the biosynthetic
production of PHAs, which have been reviewed elsewhere.173 In simplified
form, the carbon source is converted to a hydroxyacyl-CoA thioester, the
monomer which is polymerized to PHA, involving different metabolic path-
ways depending on the carbon source (Figure 6.2).
The polymerization reaction of hydroxyacyl-CoA thioester is catalyzed by
the enzyme PHA synthase, the key enzyme in the production of PHA.174 In
early investigations of the mechanism of polymerization, which was broadly
accepted in later mechanistic studies, it was proposed that two thiol groups in
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168 Chapter 6
174–177
the active sites of PHA synthase were involved in the reaction. The thiol
groups form thioesters with the monomers and a thioester–oxyester inter-
change reaction occurs, leaving one thiol group free for bonding to another
monomer. The propagation step involves bonding another monomer to the free
thiol group and a subsequent thioester–oxyester interchange reaction, adding
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Figure 6.2 Metabolic routes towards substrates for PHA synthases (reprinted from
Reference 174 with permission from Elsevier).
Scheme 6.9 Mechanism of PHA synthesis catalyzed by the two thiol groups in PHA
synthase (adapted from References 161 and 175).
View Online
carbon atom are isotactic, i.e. they are all comprised of monomers with the R-
configuration. PHA synthases usually show broad substrate specificity and by
modifying a substrate different copolymers can be obtained from a single
strain. P(3HB-co-3HV) was prepared using glucose and propionic acid as the
carbon source from Ralstonia metallidurans (formerly known as Alcaligenes
eutrophus). By using a butyric acid and pentanoic acid as the carbon source,
Ralstonia metallidurans can produce P(3HB-co-3HV) copolymers which con-
tain up to 85 mol% of HV units.180 After it was shown that Pseudomonas
oleovorans grown on octane were able to produce poly(hydroxyoctanoate),181 a
number of mclPHA were produced from this microorganism with up to 12
carbon atoms by using different sodium salts of n-alkanoic acids as sub-
strates.182,183 Substrate specificity of PHA synthases is reflected in the fact
that these enzymes can usually catalyze the polymerization of sclPHA or
mclPHA.174 However, some recent reports show that copolymers of sclPHA
and mclPHA could also be produced.184,185
A large number of strains that accumulate PHAs have been isolated.
However, only some of them are considered suitable for the production of
PHA when all factors, such as the use of an inexpensive carbon source,
growth rate, polymer synthesis rate and extent of polymer accumulation, are
taken into account.186,187 One of the most investigated is Ralstonia metalli-
durans, due to its ability to accumulate large amounts of P(3HB), up to 80%
of the dry cell mass.188,189 Alcaligenes latus and Azotobacter vinelandii were
also considered interesting for further development for PHA production
owing to their capability to produce PHA during the growth phase, which
would shorten and simplify the production process.190–192 Methylotropus were
investigated in order to develop a methanol source-based process.193,194
Pseudomonas species are of interest due to their ability to produce different
types of mclPHAs, with a great variety of functional groups introduced into
the polyester.183,195,196 In order to achieve high production of P(3HB), in
addition to wild type bacteria, recombinant types were also investigated,
especially Escherichia coli.197,198
Investigations directed toward optimization of biotechnological processes to
be performed using cheap and readily available carbon substrates are of great
importance, since the final cost of the industrial production is mainly dictated
by the cost of the carbon source. Those carbon sources which lead to struc-
turally identical monomers are designated as related, while those which are
structurally completely different from the generated monomers are classified as
unrelated. Carbon sources can vary from simple carbohydrates, alkanes and
fatty acids to plant oils and various other cheap carbon sources, such as was-
tewater from olive mills, molasses, whey, starchy wastewater, corn step liquor
and palm oil mill effluent.199
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170 Chapter 6
Synthesis of PHA in a biotechnology-based method can be performed with
batch, fed-batch and continuous cultures.199,200 Extraction of PHA from the
biomass can be performed in two ways and is an important step which
determines the final economy of a process.201 In the first approach, after
sterilization, the cells are treated with a cocktail of enzymes (including pro-
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172 Chapter 6
tougher, less brittle and more ductile polymer with improved flexibility and
impact strength is obtained. However, the susceptibility of the properties
deterioration on aging can be even worse for P(3HB-co-3HV) copolymers than
for the homopolymer. P(4HB) has a much higher tensile strength (104 MPa)
compared to P(3HB) and is a strong thermoplastic material. It also has much
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higher elongations at break, as high as 1000%, which show the high ductility of
this polymer.219 For P(3HB-co-4HB) copolymers, the tensile strength decreases
with the introduction of 4HB units up to 16 mol% to 26 MPa, and then
increases reaching 104 MPa for the P(4HB). Copolymers, P(3HB-co-4HB),
have higher elongations at break compared to P(3HB), over the whole
composition range.
The mclPHAs, compared to the sclPHAs, are polymers with lower crystal-
linity, flexible and soft and exhibit behavior like those of thermoplastic
elastomers, with low tensile strengths and high elongations at break.222–224
Copolymers of sclPHA and mclPHA, with low contents of mclPHA units, have
improved properties compared to brittle sclPHAs and exhibit properties similar
to poly(propylene).
P(3HB) and copolymers P(3HB-co-3HV) possess piezoelectricity, a property
which could be useful in medical applications for bone healing and repair
through electrical stimulation of bone pins derived from these materials.202
P(3HB) is soluble in various solvents, such as chloroform, dichloromethane
and alcohols with more than three carbon atoms. However, to completely
solubilize crystalline P(3HB) even at a low concentration, refluxing in chloro-
form for up to 24 h is required. P(3HB) is sparingly soluble in toluene, pyridine,
dioxane and octanol and is insoluble in water, lower alcohols, aromatic
hydrocarbons, diethyl ether and hexane.
P(BHB) is unstable on heating and easily decompose at temperatures above
its melting temperature with a rapid reduction in molecular weight.225 Recent
investigations on the thermal stability of P3HB and P(3HB-co-3HV) with 30
mol% of 3HV units revealed that upon continual heating in the temperature
range from 250–400 1C, the copolymer showed better thermal stability.226 Some
P(3HB-co-3HV) copolyesters (3HV=0–71 mol%) exhibited better thermal
stability on prolonged heating above their respective melting temperatures and
showed better promise for thermal processing than P(3HB), since their melting
temperatures are much lower than that of P(3HB).227 The main pyrolysis
product of P(3HB) is crotonic acid produced by the cis elimination reaction,
which also yields oligomeric products. The degradation products of P(3HB-co-
3HV) were mainly propene, 2-butenoic acid, 2-pentenoic acid, propenyl-2-
butenoate, butyl-2-butenoate and CO2.226,228
As to PCL and PLLA, the hydrolytic degradation of P(3HB) is a slow
process. A recent study showed that the weight loss of P(3HB) film in phos-
phate buffer solution at 37 1C was only 2.8% after 19 weeks of incubation.229
The hydrolysis proceeds by random chain scission with hydroxy acid oligomers
as the intermediate product and monomeric acid as the ultimate degradation
product. The rate of P(3HB) hydrolysis is affected by a number of factors such
as pH, temperature and crystallinity of the sample.151,230 The degradation
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contradictory. While some studies showed that in the range of small amounts of
3HV (12 mol%), the hydrolytic degradation of the copolymers is favored, some
studies on copolymers with higher amounts of 3HV units (45 and 71 mol%)
found a decreasing hydrolysis rate.229,231 As proposed by Albertsson et al.,
there might be some maximum in the 3HV content above which the hydrolysis
is no longer favorable.232
PHAs are readily degraded in the presence of enzymes PHA depolymerase.
Since PHAs are not water soluble and, as solids, can not be directly absorbed by
microorganisms, many of them excrete specific enzymes PHA depolymerase.
These enzymes degrade PHAs extracellulary to form oligomeric products which,
being soluble and of low molecular weight, can easily enter a cell, where they are
further metabolized. Enzymatic degradation of PHAs occurs at the surface of
the polymer, where the enzymes are absorbed via a binding domain and are
further involved in the PHA hydrolysis via a catalytic domain. The enzymatic
degradation commences in the amorphous regions of the polymer specimen,
while in the later stages both amorphous and crystalline phases are degraded
without preference. Thus, the degree of crystallinity and crystallite size have a
profound effect on the rate of degradation.221,233,234 Some studies showed that
an atactic, completely amorphous P(3HB) obtained by a chemical route is not
prone to enzymatic attack and that the biodegradation can be induced by the
presence of some crystalline phase. For example, natural P(3HB), which can
provide appropriate enzyme binding sites, was shown to induce the degradation
of amorphous P(3HB).235 The substrate specificity of the catalytic domain of
PHA depolymerases, investigated on the number of different aliphatic polye-
sters, was shown to be relatively narrow.236 With the introduction of a second
co-monomer unit, 3HA, the rate of degradation of the copolymers was
increased by 5–10 times compared to P(3HB), however, only up to 10–20 mol%
of the comonomer units.224,237–239 A further increase in the co-monomer content
decreased the degradation rate. It was also shown that an increase in the
side-chain length at the b-carbon for copolymers with larger fractions of long
HA units, further reduced the rate of ester bond hydrolysis by the enzyme.240
Microorganisms that can degrade PHAs are widely distributed in different
environments. The percentage of P(3HB)-degrading microorganisms in the
environment was estimated to be 0.5–9.6% of the total colonies.241 Many of
them, belonging to bacteria and fungal strains, have been isolated and inves-
tigated.187 PHAs completely degrade to CO2 and water in an aerobic envir-
onment and to methanol under anaerobic conditions. The degradation of
PHAs was investigated in different environments, such as soil, sea water,
compost and sewage sludge.151,242–246 Complete degradation of P(3HB-co-
3HV) copolymer was shown to occur in 6, 75 and 350 weeks in anaerobic
sewage, soil and sea water, respectively.186
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174 Chapter 6
P(3HB) is also considered for medical applications. b-Hydroxybutyric acid,
as a product of P(3HB) hydrolysis, can enter the metabolic pathways of
organisms since it is a normal constituent of human blood. Degradation of
PHA in vivo is slow due to the lack of suitable enzymes (PHA depolymerases).
Some investigations on the fate of P(3HB) implants in mice have shown that the
weight loss of the sample was 1.6% 6 months after implantation.247 In terms of
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176 Chapter 6
Another approach to increase the molecular weight of polyesters obtained in
direct polycondensation is to use chain-extenders, for example through the
reaction of a,o-functionalized polyesters with diisocyanates or oxazo-
lines.254,267,268 Using hexamethylene diisocyanate as a chain extender, an
increase in the number average molecular weight from 33 000 to 72 000 g mol 1
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for copolymers with butylene succinate and butylene adipate units was
reported.267
As previously stressed, the advantage of the polycondensation reaction is the
variety of monomers (aliphatic, aromatic, cyclic) which can be introduced into
the polyester chain without great alteration of the reaction conditions. For
example, segmented poly(ether ester)s, based on PBS hard segments and with
polyethers in soft segments, used to increase hydrophilicity of copolyesters,
were successfully obtained through transesterification polycondensation using
Ti(OBu)4 as a catalyst and the corresponding polyether macrodiols. Different
polyethers such as poly(ethylene oxide) (PEO), poly(propylene oxide) (PPO) or
poly(tetramethylene oxide) (PTMO), could be used as polyether component in
the soft segments (Scheme 6.11).269–272 Using the same reaction procedure,
unsaturated groups through the reaction of dimethyl fumarate could be suc-
cessfully incorporated into the PBS polyester chains (Scheme 6.12).273
The synthesis of polyesters by enzyme-catalyzed reactions are gaining con-
siderable interest.274–276 The milder reaction conditions, the absence of a toxic
metal catalyst and the possibility of having good control over the polymer
structure are obvious advantages of such a reaction pathway to polyesters.
Simultaneously, the low molecular weights of the products and high cost of
enzymes are disadvantage of such processes, indicating the need for further
improvements in enzyme-catalyzed syntheses for the production of suitable
products.
Scheme 6.11 Structural formula of poly(ester ether)s with PBS as the hard segments
and different polyethers in soft segment: PEO (R = H, x = 1), PTMO
(R = H, x = 3) or PPO (R = CH3, x = 1).
178 Chapter 6
non-biodegradable polymers such as LDPE or PP. Since the mechanical
properties are very dependant on the crystallinity, these properties usually
decrease with copolymerization. The decreasing trend in mechanical properties
usually follows the decrease in the crystallinity with the incorporation of units
of the second comonomer. This was observed for a number of copolyesters of
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the introduction of cyclic units into an aliphatic polymer backbone can greatly
increase thermal stability of random copolyesters.284,295,296 As expected, the
introduction of thermally labile ether bonds into polyesters deteriorates the
thermal stability of the copolyesters.292
Hydrolytic degradation of PBS is slow, due to its hydrophobic nature and
high crystallinity. It was reported that the residual mass of a PBS sample after
incubation in a phosphate buffer solution for 15 weeks was 35%.297 Hydrolytic
degradation of PBS proceeds with an immediate and continuous decrease in the
molecular weight, which is followed by weight loss in the later stages of
degradation. This suggests a homogenous degradation mechanism similar to
that of other aliphatic polyesters. The degradation rates are increased in more
alkaline solutions.252 The degree of crystallinity has a major influence on the
degradation rate, since the degradation commences first in the amorphous parts
of the polymer. In addition to crystallinity, the internal structure of the PBS
crystallites also influence the progress of hydrolysis.298 Other types of aliphatic
polyesters exhibit the same tendencies. For copolyesters, the interplay between
the usually reduced crystallinity of copolyesters and the chemical composition
dependencies will result in an increased or decreased rate of hydrolysis for
certain copolyester compositions.252
The degradation of poly(alkylene dicarboxylate)s is enhanced by lipase
enzymes.256,299,300 Lipases are water soluble and act on insoluble substrates in
an effective manner only on an oil–water interface.301 The enzymatic degra-
dation rate depends on a number of factors such as chemical composition,
hydrophylicity/hydrophobicity balance, degree of crystallinity, size of spher-
ulites and lamellar thickness. The degradation rate also depends on external
factors, such as the employed enzyme, temperature and pH at which the
experiment is performed. It would be reasonable to expect that the polyesters
with higher concentration of ester linkages within the polymer chain would
exhibit higher rates of degradation, but this is only observed in some cases.299
Moreover, by increasing the hydrophilicity of a polyester chain through the
introduction of hydrophilic polyethers, e.g. PEO, the degradation of polyesters
is enhanced.269,270 However, the higher order structure directed by particular
chemical structures seems to have greater influence on the degradation and
often screens the chemical composition dependence. The degree of crystallinity
is the factor which predominately determines the degradation rate in an
unfavorable fashion. This is the main reason for usually observed higher rates
of degradation for copolyesters compared to homopolyesters.256,280,301 It was
shown that chain mobility, which is correlated with the melting temperature
of polyester, has a significant influence on the rate of enzymatic degradation.
A dependence of degradation rate of polyesters on the temperature difference
between the melting temperature of the polyester and temperature of
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180 Chapter 6
302
experiment (DTmt) was postulated by Marten et al. These authors correlated
DTmt with chain mobility in the crystalline domains of the material, the
degradation of which is the rate-determining step in an enzymatic degradation
process of crystalline polyesters. This concept also applies to aliphatic–
aromatic copolyesters, which showed reduced degradability as a consequence
of more rigid chain structures.303–305
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182 Chapter 6
importance as a solution to solid waste management and is often the first
option to be considered. However, many polymers can not be easily separated
for recycling or can not be recycled at all. In addition, the recycling of poly-
meric materials can not be repeated indefinitely. In such cases, biodegradable
polymers are good alternative. In nature, the biodegradation of polyesters
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Melting temparature, 150–165 60–62 170–175 114–115 93–95 110–115 108 255
1C
Glass transition 55–65 –60 –32 –45 –120 78
temperature, 1C
Density, g cm 3 1.25 1.20 1.26 1.23 1.25–1.27 0.92 1.4
Tensile yield 48 17.5 32 34 19 35/44 12 58
strength, MPa
Flexular modulus, 3828 411 2200 580 340 95/80a 176 2900a
MPa
Biodegradable Polyesters: Synthesis and Physical Properties
184 Chapter 6
6.6.2 Medical Applications
Biodegradable polymeric materials comprise a large group of biomaterials,
i.e., materials intended to interface with biological systems. Whenever a
biomaterial is required for a limited period, the use of polymers which can
biodegrade is beneficial, since there is no need for surgical removal of the
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CHAPTER 7
7.1 Introduction
Petrochemical polymers have become ubiquitous for their excellent properties
and durability. Unfortunately, they also create an enormous environmental
burden. Motivations behind sustained research in reducing dependence on
polymers from petrochemical sources are similar to those in energy research; a
decreasing fossil fuel supply with a corresponding price increase and a wide-
spread awareness of sustainability.1 As a result, finding new uses for agri-
cultural commodities has become an important area of research.
Petroleum-based materials could potentially be replaced with renewable and
biodegradable materials such as polysaccharides or proteins.2 Biodegradable
materials are capable of undergoing microbial or enzymatic degradation,3 although
this does not does not necessarily imply renewability, and several petroleum-based
biodegradable polymers are commercially available. Biodegradable polymers are
generally classified as outlined in Figure 7.1.3–5 In some cases blends between
natural and synthetic polymer are considered a separate class.6
The potential use of agro-polymers in the plastics industry has long been
recognized. Agro-polymers are extracted from either plants or animals. They
could contribute to a reduction of dependence on fossil resources as agri-
cultural resources are generally sustainable. Some of the polymers in this family
197
198 Chapter 7
Biodegradable polymers
Other
Renewable polymers
7.1.1 Polysaccharides
Polysaccharides are carbohydrate polymers, formed from either mono- or di-
saccharides through glycosidic bonds. The most significant polysaccharides of
relevance here are cellulose, starch, lignin, pectin and chitin. The structures of
some of these polymers are shown in Figure 7.2.
Cellulose is one of the most abundant biopolymers and forms the major
constituent of plant cell walls. Currently cellulose is only processed thermo-
plastically after chemical modification;2 cellulose acetate would be the most
significant product of this nature.
Starch is produced by many plants as a source of stored energy and is found
in plant roots, stalks, crop seeds and crops such as rice, corn, wheat, tapioca
and potato. It consists of linear amylose and branched amylopectin.2 Starch is
recovered by wet grinding, sieving and drying as native starch.7
Lignins are aromatic amorphous polymers and are obtained from almost all
types of natural plants, in association with cellulose.8 Lignins are recovered
Synthesis and Characterization of Thermoplastic Agro-polymers 199
A B
7.2 Synthesis
It is well known that polymers can generally be classified as either thermoset,
thermoplastic, or rubbery. Most agro-polymers do not behave thermo-
plastically without some additives and would typically degrade before a flow-
able melt can be formed. It is therefore necessary to consider the synthesis route
to render these materials ready for thermoplastic processing.
Polymer processing is concerned with the mixing and shaping of polymeric
materials to form them into useful products. Processing usually involves the
application of heat and pressure, with the specific method depending on whe-
ther the material is thermoplastic or thermoset.10 Thermoplastic processing
involves melting a polymer, followed by shaping and finally cooling the
material in its new form. The softening temperature and atmospheric stability
of the polymer, as well as the geometry and size of the finished product, are
important. The heat required for melting can be supplied by radiation, con-
duction, or mechanical work. The most important thermoplastic processing
techniques are extrusion, post-die processing, thermo-forming and injection
moulding. The largest volume of thermoplastics is probably processed by
means of extrusion.10
An extruder consists of a heated, fixed metal barrel that contains either one
or two screws. The screws act by conveying the material through the heated
barrel, inducing shear forces and increasing pressure along the barrel before
Synthesis and Characterization of Thermoplastic Agro-polymers 201
leaving the extruder at the die. Process variables include the feed material’s
composition, screw speed, barrel temperature profile, feed rates and die size and
shape. The degree of screw fill, specific mechanical energy input (SME), torque,
pressure at the die, residence time and product temperature are influenced by
these process variables.11
7.2.3 Starch
One specific thermoplastic agro-polymer of interest here is thermoplastic starch
(TPS). Starch can only be converted into a thermoplastic material in the
presence of plasticizers using heat and shear.1,6 The benefit of TPS is its
renewability, compostability and relatively low price compared with synthetic
thermoplastics.8 One problem with the use of starch is its high glass transition
temperature (Tg). Brittleness also increases with time due to free volume
relaxation and retrogradation. In order to increase flexibility and processa-
bility, plasticizers such as water, glycol, sorbitol, urea, amide, sugars and
quaternary amines have been used in TPS.6 Dimensional stability and
mechanical properties of thermoplastic starch are highly dependent on moist-
ure content.1 Unfortunately, the excessive hydrophilicity of starch is not
significantly reduced by polyol plasticizers.
In its native state, starch is semi-crystalline (about 20–45%) and water
insoluble. Native starch granules typically have dimensions ranging from 0.5
to 175 mm and appear in a variety of shapes. It is composed of linear
(amylose) and branched (amylopectin) polymers of a-D-glucose. Amylose
has a molecular mass of about 105–106 g mol1, while amylopectin has a
molecular mass in the range 107–109 g mol1. Starch rich in amylose is
usually preferred for conversion to TPS as the linearity of amylose improves
the processability of starch even though it is present as a minor component
(between 20 and 30wt%). The ratio of amylose to amylopectin depends on
the source and age of the starch, and can also be influenced by the
extraction process.4,9,13,14
Synthesis and Characterization of Thermoplastic Agro-polymers 203
TPS is obtained after disruption and plasticization of native starch’s granular
structure into a homogeneous amorphous phase by applying thermo-
mechanical energy in a continuous extrusion process. Water and glycols are
commonly used plasticizers, although urea and formamide have also been
explored.15,16 A chemical agent, such as urea, favours granule destruction by
disrupting hydrogen bonding in the crystallites.9 Thermal processing of TPS
involves multiple chemical and physical reactions:13
water diffusion
granule expansion
gelatinization
decomposition/destruction
melting and crystallization.
7.2.4 Proteins
It has been said that in order for proteinaceous bioplastics to be commercially
feasible, they need to be processable using equipment currently used for syn-
thetic thermoplastics. Proteinaceous bioplastics are often brittle and water
sensitive, and overcoming this is one of the driving forces behind research in
this field. Physiochemical properties and processing conditions are often gov-
erned by the protein’s structural properties, and therefore also final material
properties.17
A synthetic polymer consists of identical monomers, covalently bonded in a
long chain. Unlike synthetic polymers, proteins are complex hetero-polymers,
consisting of up to 20 different amino acid repeat units. The amino acid repeat
unit contains two carbon atoms as well as nitrogen, differing only in their
functional side groups. In its natural environment, a protein will be folded into
secondary, tertiary and quaternary structures stabilized through hydrophobic
interactions, hydrogen bonding and electrostatic interactions between amino
acid functional groups. The folded conformation is a delicate balance of these
interactions.18 Once folded, the structure may be stabilized further with strong
covalent cross-links. Due to the diverse building blocks of proteins and their
unique structure, a large variety of biodegradable materials can be produced
offering a wide range of functional properties.11
It is well known that the visco-elastic behaviour of amorphous or semi-
crystalline polymers can be divided into five regions; these are the glassy,
leathery, rubbery rubbery-flow and viscous states. The transformation from
one region to another is dependent on temperature, while the temperature at
which each transition occurs is dependent on the polymer structure. Processing
can only be done above temperatures corresponding to the rubbery-flow
region. Most literature on proteinaceous bioplastics suggests that processing be
done above the protein’s softening point, which would imply a temperature well
above the Tg.
Extrusion and injection moulding of polymers require that a viscous melt be
formed by the polymer upon the addition of heat. That implies that interactions
Synthesis and Characterization of Thermoplastic Agro-polymers 205
between chains are sufficiently low to allow relative movement of chains, but
some interaction is required to impart some degree of melt strength in the
material. Synthetic polymers generally satisfy these requirements. In the
absence of strong intermolecular forces (hydrogen bonds), chain entanglements
and van der Waal’s forces are the most important mechanisms that impart a
good melt strength.
Small changes in environmental conditions, such as increasing temperature,
pressure, change of pH or addition of chemicals, can disrupt a protein’s folded
conformation and this is called denaturing. Denaturation is a unique property
of proteins and can be defined as the modification of secondary, tertiary or
quaternary structures of a protein molecule. This is not to be confused with
degradation, which is the loss of primary structure, or breaking of covalent
peptide bonds. For a protein to behave like a synthetic polymer, the protein
chain is required in an extended conformation enabling the formation of
sufficient chain entanglement. In order to do this, multiple non-covalent and
covalent interactions need to be reduced, allowing chains to unfold and form
new interactions and entanglements.10,11
Unlike starch, where destructuring leads to an amorphous fluid, denatura-
tion of proteins exposes core structural groups which can be more hydrophobic
than surface groups (depending on the environment). In a polar environment
the structure forms with polar residues in the surface and hydrophobic ones
inside, and vice versa. Furthermore, with an increase in temperature, chains
become more mobile but their movement is restricted because of newly formed
intermolecular forces. Hydrophobic interaction intensifies due to temperature
and denaturation which is followed by coagulation.9
Hydrogen bonds, ionic interactions, hydrophobic interactions and covalent
disulfide bonds are affected during denaturation, causing an unravelling of
protein structure into a random coil conformation. Denaturation can have
several important consequences, such as increase in viscosity of protein solu-
tions, decrease of solubility due to exposure of hydrophobic groups, increase of
reactivity of side groups, altered sensitivity to enzymatic proteolysis and altered
surfactant properties. Denaturing exposes functional groups of the amino acid
side chains, thereby introducing new interactions by means of hydrophobic,
hydrogen or ionic bonding. Although some of these effects can be seen as
negative, denaturation and the consequences thereof are important for protein
processing.
Understanding how to process agro-polymers with similar properties to
synthetic polymers requires an understanding of how these forces are mani-
fested in proteins. The processability of proteins depends on their transition
from the glassy to rubbery and viscous-flow states. These transitions are
achieved with judicial application of heat, pressure, shear, chemical additives
and plasticizers. Specific amino acid residues (primary protein structure) and
initial structure (natural protein state) of the protein will influence each of
these factors. Based on the structure of proteins and the requirement for
thermoplastic processing, three broadly categorized processing requirements
have been identified:11
206 Chapter 7
breaking of intermolecular bonds (non-covalent and covalent) that sta-
bilize proteins in their native form by using chemical or physical means
arranging and orientating mobile chains in the desired shape
enabling formation of new intermolecular bonds and interactions to sta-
bilize the three-dimensional structure.
7.3 Characterization
7.3.1 Overview of Characterization
Selecting appropriate biopolymers for commercial use is a complicated task
and involves careful consideration of the properties of the polymeric material.
In the context of this chapter, the meaning of the term ‘Characterization
of Agro-polymers’ is twofold. Not only are the techniques used for
Synthesis and Characterization of Thermoplastic Agro-polymers 207
characterization important, but also how they define the characteristics of agro-
polymers. Typically, chemical structure and morphology as well as mechanical
and thermal properties are important. In addition to these, aspects that need
consideration are the shape and form of raw materials as well as their quality,
supply, cost and physical properties.26 Obtaining optimal material properties in
a final product requires appropriate processing which is dependent on struc-
ture. Structure, property and processing cannot be considered independently,
as illustrated in Figure 7.3.
The structure of polymers is described at multiple levels. Biochemists tradi-
tionally refer to the primary, secondary and tertiary structure of native pro-
teins. Similarly for thermoplastics there are multiple levels of structural
information, such as chain architecture and crystallinity. All of these contribute
to observed material properties and can be manipulated by processing. In
Figure 7.4, the general structure of proteins, starch and synthetic polymers are
compared. At the monomeric level starch is made up of repeating units of
glucose and exists in two forms – branched (amylopectin) or unbranched
(amylose). In native starch these are arranged in granules consisting of hard
and soft regions.27 The blockets in the hard shell are made up of crystalline
amylopectin surrounded by amorphous amylose. Processing disrupts these
ordered regions.28 Proteins consist of a chain of peptide linkages with various
amino acid side groups present. These side groups contribute to a range of
interactions which, combined with hydrogen bonding around the polypeptide
backbone, lead to different secondary structures.11 Synthetic plastics include a
range of materials with different monomers and linkages. Olefins, or vinyl-like
polymers, such as polypropylene are hydrophobic, whereas nylons contain
amide linkages similar to the peptide backbone in proteins which are hydro-
philic and form hydrogen bonds with water.29 Vinyl-type polymers form helical
conformations similar to a-helices in proteins and many petroleum-derived
Structure Processing
Monomer’s chemical structure Plasticization
Inter and intramolecular interactions Viscosity and rheology
Crystalline and amorphous content
Morphology
Figure 7.4 Monomers and microstructure of starch, protein and examples of synthetic thermoplastics. Microstructure of starch reprinted from Car-
bohydrate Polymers, 32(3–4), D. J. Gallant, B. Bouchet, and P. M. Baldwin, Microscopy of starch: Evidence of a new level of granule
Chapter 7
organization, page No. 188, Copyright (1997), with permission from Elsevier.27 Gelatinization and retrogradation of starch reprinted with
kind permission from Springer Science+Business Media: Journal of Materials Science Microstructure and mechanical properties of
orientated thermoplastic starches, 40(1), 2005, 115, L. Yu and G. Christie, figure 4, r 2005 Springer Science + Business Media, Inc.28
Synthesis and Characterization of Thermoplastic Agro-polymers 209
30,31
polymers are semi-crystalline and contain spherulites. Within these there
are crystalline regions surrounded by an amorphous region with similar
morphology to the amylopectin clusters in starch.29 The techniques discussed in
this section are used to assess these structural aspects and how they define the
characteristics of thermoplastic starch or proteins.
Techniques such as Fourier transform infrared spectroscopy (FT-IR) and
nuclear magnetic resonance (NMR) are used in determining information about
the chemical structure of the monomers and the nature of covalent bonds
between them. Molecular mass and molecular mass distribution, as well as
chemical nature of side groups, determine the interaction between polymer
chains. Where interactions between chains lead to ordered regions, crystalline
phases are observed, whilst other less ordered regions are said to be amorphous.
X-ray diffraction is often used to assess structural information, such as the
degree of crystallinity and specific crystal structures while microscopy techni-
ques, such as SEM or TEM, are used to determine morphology.
All these different levels of structure contribute to the quality of the final
product, as defined by it mechanical and physical properties. Important
mechanical and physical properties are summarized in Table 7.1.26
As well as being rate dependent, the mechanical properties of polymers
show significant temperature-related effects. Of particular importance for
Linear,
with strain
Non-linear
hardening
Rubber-like
Elongation (%)
A B
Figure 7.7 Fracture surface of SPI plasticized with glycerol, with (B) and without (A)
stearic acid.53 Reprinted from Industrial Crops and Products, 21(1),
P. Lodha and A. N. Netravali, Thermal and mechanical properties of
environment-friendly ‘green’ plastics from stearic acid modified-soy
protein isolate, 49–64, Copyright (2005), with permission from Elsevier.
A B C
Figure 7.8 Tensile fracture surfaces of (A) glycerol, (B) sorbitol and (C) polyester
amide plasticized soy-based agro-polymers.48 Reprinted with permission
from P. Tummala, W. J. Liu, L. T. Drzal, A. K. Mohanty and M. Misra,
Ind. Eng. Chem. Res., 2006, 45, 7491–7496. Copyright 2006 American
Chemical Society.
In a further example, the tensile fracture surfaces for soy protein plasticized
by glycerol, sorbitol and polyester amide are shown in Figure 7.8. It can be seen
that glycerol plasticized samples showed ductile fracture features with a coarse
surface. Sorbitol plasticized samples showed brittle fracture features with
relatively smooth surfaces. These samples also had higher strength and stiff-
ness, but lower elongation at break. Using polyester amide as a plasticizer
resulted in local ductile fracture features and the samples had moderate
mechanical properties.48
If soy protein is plasticized with acetamide, it was shown that brittle fracture
dominated up to 20% acetamide, as evident from the sharp ridges on the SEM
images (Figure 7.9). As plasticizer content increased the surface become
214 Chapter 7
Figure 7.9 SEM images of cross sections of soy plastic sheets plasticized with different
amounts of acetamide.54 Liu, D. and L. Zhang: Structure and properties
of soy protein plastics plasticised with acetamide. Macromolecular Mate-
rials and Engineering 2006, 291, 820–828. Copyright Wiley-VCH Verlag
GmbH & Co. KGaA. Reproduced with permission.
smoother, but above 30% the surface again appeared fluctuant, most likely
because of excess plasticizer.54
Similar observations have been made for starch plasticized with glycerol,
xylitol, sorbitol or maltitol. Fractured surfaces are shown in Figure 7.10 and it
was observed that the surface of glycerol plasticized material (A) was rough.
This was likely due to glycerol- and amyl pectin-rich domains forming a het-
erogeneous material. The fractured surface of xylitol plasticized system (B)
appeared different with a rough structured surface, while sorbitol (C) and
maltitol (D) plasticized surfaces were perfectly smooth.55
Unfortunately, overall mechanical property improvements in agro-polymers
have been slow. A large range of starch-based plastics have been surveyed in
terms of strength at break versus their elongation at break.15 It has been shown
that the mechanical properties follow a master curve that is almost exclusively
dependent on the amount of plasticization (Figure 7.11).15 The situation for
protein-based plastics is not much different, and tensile strengths seldom exceed
25 MPa, with elongation at break being less than 140%.35–38,41,42,56 As with
starch-based materials, this behaviour is mostly determined by the amount of
plasticizer. Very little research has been successful in developing high strength
agro-polymer, with corresponding high elongation.
Synthesis and Characterization of Thermoplastic Agro-polymers 215
Figure 7.10 Scanning electron micrographs of the fractured surface of (A) glycerol,
(B) xylitol, (C) sorbitol and (D) maltitol plasticized starch. Reprinted
with permission from A. P. Mathew and A. Dufresne, Biomacromole-
cules, 2002, 3, 1101–1108. Copyright 2002 American Chemical Society.
Figure 7.11 Tensile strength versus elongation at break for a variety of starch based
agro-polymers.15 N. Follain, C. Joly, P. Dole and C. Bliard: Mechanical
properties of starch-based materials. I. Short review and complementary
experimental analysis. Journal of Applied Polymer Science, 2005, 97(5),
1783–1794. Copyright John Wiley and Sons. Reproduced with
permission.
216 Chapter 7
7.3.2.1 Factors Affecting Mechanical Properties
Moisture Content. The moisture content or water uptake of agro-polymers is
an important topic and just about every research paper dealing with agro-
polymers also considers moisture content.44,55,57–63 Moisture content is
important from at least two perspectives. Most importantly, the moisture
content directly influences the mechanical properties of the plastic, typically
increasing elongation and reducing strength by effectively plasticizing the
material.4,10,11,44 Secondly, in protein-based systems, the ability to absorb water
could also be an indication of cross-link density.10,59
Water is required in the manufacture of both thermoplastic starch and
thermoplastic protein. For starch, it ensures gelatinization and the formation of
a continuous thermoplastic phase. In proteins, it lowers the glass transition and
denaturing temperature,10,11 allowing processing. However, dry resin ensures
dimensional stability directly after injection moulding and over time.
The equilibrium moisture content of hydrophilic agro-polymers depends on
the relative humidity of the environment and is typically described by the
Guggenheim-Anderson-de Boer (GAB) and Brunauer-Emmett-Teller (BET)
equations.63,64 Equilibrium moisture content is also influenced by the type and
amount of plasticizer in the system. Careful selection of additives and plasti-
cizers could control the equilibrium moisture content of these materials, but
only to a limited extent. The equilibrium moisture content of starch as a
function of relative humidity and plasticizer type and content has been deter-
mined. It was found that the equilibrium moisture content for starch can be as
high as 30% at 70% relative humidity, but, by using different plasticizers, this
number could be significantly reduced.55,62 In Figure 7.12, a phase diagram for
a water/glycerol/starch system (as a function of plasticizer content) is shown
which highlights the importance of the interactions between water, plasticizer
and polymer.65
DSC. DSC measures the difference in heat flow between a specimen and a
reference as a function of temperature. Specimens are placed in a special pan,
which can either be sealed or not. The reference is usually an identical, but
Figure 7.14 Identification of the glass transition as a step change in heat flow,
CA ¼ citric acid, CA0 ¼ TPS with 30 pph glycerol, CA1 ¼ TPS with 30
pph glycerol and 10 pph citric acid, CA2 ¼ TPS with 30 pph glycerol and
20 pph citric acid, CA3 ¼ TPS with 30 pph glycerol and 30 pph citric
acid, CA4 ¼ TPS with 30 pph glycerol and 40 pph citric acid.70 Reprinted
from Carbohydrate Polymers, 69(4), R. Shi, Z. Z. Zhang, Q. Y. Liu,
Y. M. Han, L. Q. Zhang, D. F. Chen and W. Tian, Characterization of
citric acid/glycerol co-plasticized thermoplastic starch prepared by melt
blending, 248–755. Copyright (2007), with permission from Elsevier.
Synthesis and Characterization of Thermoplastic Agro-polymers 221
Table 7.3 Typical DSC responses to thermal events in polymeric
samples.78,79
Effect on plot of heat
Thermal Event flow vs temperature
It would be better to measure the glass transition by cooling, but for his-
torical reasons, it is usually measured in heating.79 The typical DSC responses
to this and other thermal events are listed in Table 7.3.
DSC is sensitive to the thermal history of the sample. A common approach is
to heat and cool a specimen prior to scanning for thermal events during a second
heating cycle. The first heating scan provides information about the thermal
history (processing or ageing) of the sample. The cooling and second heating
cycle are then performed at known thermal history. Transitions, such as the glass
transition, are then determined from the results of the second scan.80 This may
pose a concern for proteins as heating during the first scan may affect protein
conformation and hydration. An alternative approach is to use modulated DSC,
in which the heating rate oscillates.81 This allows separation of reversible events
(glass transition and melting or fusion) and non-reversible events (oxidation,
curing, relaxation and cold crystallization) which is complicated as these may
overlap.78,81 For starch this can be particularly useful for studying the multiple
transitions that occur during gelatinization, although the onset and peak
temperatures appear lower than those observed in conventional DSC.82
One other complication is that results from a DSC experiment depend on the
rate of temperature change. For this reason the temperature ramp rate should
always be reported with the results. It is also advisable to complement the
analysis with other techniques, such as DMA.
the elastic (or storage) modulus (E’), representing the elastic component of
the material’s response, or energy stored by reversible deformation of the
material
the loss modulus (E’’), representing the viscous component of the mate-
rial’s response, or energy dissipated as heat by molecular rearrangement
222 Chapter 7
the damping coefficient (tan d); this is the tangent of the phase lag between
applied force and the sample responding and is equal to E’’/E’.
The glass transition region is identified as the region in which the elastic
modulus drops rapidly by around two orders of magnitude and there is a local
maximum in the loss modulus. These contribute to a clear peak in the plot of
tan d versus temperature.14,48,80,84–86 The glass transition temperature could
also be reported as the peak in the loss modulus or the onset of drop in storage
modulus.47,87
DMA results for the glass transition are frequency dependent. For this
reason the frequency of testing should always be reported. It is also good
practice to test at more than one frequency and compare results with those
from other techniques, such as DSC. For agro-polymers, the loss of moisture
during scanning may also contribute to higher observed glass transition tem-
peratures than those determined using DSC in sealed pans.58
Below the Tg, other thermal transitions relating to short range movements
within polymer chains are identified as regions of rapid drop in storage mod-
ulus, but these are less dramatic than the glass transition. Above the glass
transition, protein denaturing has been associated to a minimum in tan d.88
Figure 7.15 shows examples of plots of tan d versus temperature for a ther-
moplastic protein and a thermoplastic starch. For the soy protein, b transitions
can clearly be seen, along with a peak for ice melting at high water contents.
The low temperature peak observed for the amylose films is likely due to the
glycerol-rich phase, whereas the shifting upper peak will be the glass transition
of the plasticized amylose.
0.5 0.5
Soy protein Glycerol plasticized amylose films
tanδ
tanδ
0.25 0.25
0 0
−100 −50 0 50 100 150 −100 −50 0 50 100 150 200
Temperature (°C) Temperature (°C)
Figure 7.15 Plots of tan d versus temperature for DMA scans of extruded soy protein
sheets at different moisture contents58 (reprinted from Polymer, 42(6),
J. Zhang, P. Mungara and J. Jane, Mechanical and thermal properties of
extruded soy protein sheets, 2569–2578. Copyright (2001), with permis-
sion from Elsevier) and amylose films plasticized with different glycerol
contents89 (reprinted from Carbohydrate Polymers, 22(3), G. K. Moates,
T. R. Noel, R. Parker and S. G. Ring, Dynamic mechanical and
dielectric characterisation of amylose-glycerol films, 247–253. Copyright
(2001), with permission from Elsevier.)
Synthesis and Characterization of Thermoplastic Agro-polymers 223
TGA. In TGA mass loss is measured as a function of temperature. The
sample is subjected to both a controlled temperature profile and a controlled
atmosphere.79 This reveals information such as the temperatures where
moisture, solvents and plasticizers are volatilized from the material, as well
as mass loss due to combustion or pyrolysis. Many instruments offer com-
bined SDT analysis which allows for easier identification of the cause of
mass loss events.78 Additionally, FT-IR analysis can confirm volatilized spe-
cies and decomposition products associated with mass loss events.86 One of
the most common applications of TGA is determination of thermal stability
by heating to high temperatures in an inert atmosphere, usually nitrogen.79 If
a polymer is to be used in air, it is also important to determine its thermo-
oxidative stability.79 An example of the output of a TGA experiment is
shown in Figure 7.16 for soy protein isolate with plasticizer made up of dif-
ferent ratios of e-caprolactone to glycerol in both air and an inert atmo-
sphere. The mass loss is usually presented as a function of temperature, and
the first derivative is also plotted against temperature to more clearly deter-
mine the onset of thermal events.
For characterization of new materials it is advisable to utilize TGA prior to
techniques such as DSC or DMA where thermal decomposition should be
avoided.
Figure 7.16 Percentage mass loss and first derivative TGA thermograms of SPI with
40 wt% plasticizer containing e-caprolactone/glycerol ratios of 0:8 (C0),
2:6 (C2), 4:4 (C4), 6:2 (C6) and 8:0 (C8) in N2 (left) and air (right).86
Reprinted with permission from P. Chen, H. Tian, L. Zhang and P. R.
Chang, Ind. Eng. Chem. Res., 2008, 47, 9389–9395. Copyright 2008
American Chemical Society.
224 Chapter 7
Other Techniques. Differential temperature analysis (DTA) is similar to
heat flux DSC. The difference in temperature is reported, but not converted
into heat flow. This gives a similar shaped curve, but only provides qualita-
tive information rather than the quantitative information available from
DSC. Its main advantage is when run simultaneously to TGA, it can provide
insight into the cause of mass loss events; e.g. exothermic (i.e. burning) or
endothermic (i.e. evaporation). Additionally, DTA instruments can operate
over a wider temperature range than DSC.78 Although this may be an
advantage for thermal analysis of some materials (e.g. metals) that melt at
high temperatures, the relatively low degradation temperature of agro-
polymers falls within the range of a typical DSC instrument.
In thermo-mechanical analysis (TMA) the change in dimensions under a
minimal load is measured as a function of temperature.83 This reveals the
coefficient of thermal expansion (CTE) and can also be used to detect other
transitions, such as the glass transition.
The heat deflection temperature (or heat distortion temperature) is an
important material property mostly used to determine a material’s useful
temperature operating range. It refers to the temperature below which a
moulded object can hold its own shape. It can be determined using a dynamic
mechanical analyser set to apply a constant force. The HDT was determined
for blends of plasticized soy flour (52% protein) and polyamide (nylon) as 45 1C
when plasticized with 20 wt% sorbital, 35 1C when plasticized with 20 wt%
glycerol, and 39 1C when plasticized with 10 wt% of each.48 When such a blend
is used to make composites with natural fibres, increasing content of natural
fibres also increased the HDT.
Phase transition analysis uses an apparatus similar to a closed-chamber
capillary rheometer. It measures displacement at a constant pressure. It yields
glass transition results consistent with DMA, DSC and capillary rheometry.80
7.3.3.6 FT-IR
FT-IR is a technique used to investigate changes in the nature of chemical
bonds. Different covalent interactions stretch, vibrate or bend at specific fre-
quencies and allow the identification of specific interactions. With the exception
of exact optical isomers, the same IR spectrum will not be observed for com-
pounds with different structures.111 Complete determination of the cause of
every peak and elucidation of the exact chemical structure is not practical for
mixtures of complex macromolecules. Rather, changes in the location or
magnitude of characteristic peaks provide information about structural and
chemical changes occurring during processing. Important changes after ther-
moplastic processing investigated by FT-IR include changes in secondary
structures (a-helices and b-sheets or turns) in proteins and the interaction
between chains and plasticizers in both starch and proteins.
using dried protein powders in potassium bromide (KBr) pellets.113 Plastic films
may be mounted directly in FT-IR spectrophotometers.
Although the native secondary structure is denatured during thermoplastic
processing, secondary structure elements may contribute to physical fixation as
the processed material cools.97 Changes to secondary structure induced by
thermoplastic processing depend on the protein’s primary structure, processing
conditions and additives such as plasticizers or denaturing agents. In general,
extrusion appears to favour an increase in ordered b-sheet regions at the
expense of a-helices, but this is not always the case.11 Time resolved FT-IR of
soybean protein films held at 100 1C showed changes in the amide I region over
time that may be attributed to an increase in b-turn or weak b-sheet struc-
tures.114 The peaks in the amide I region overlap and some form of deconvo-
lution is needed to distinguish the combined peak into separate peaks relating
to different secondary type structures, as shown in Figure 7.17.
The amide I region for hot pressed films of egg albumin, lactalbumin, feather
keratin and wheat gluten were analysed with deconvolution software. Increased
order was observed in the form of b-sheets as glycerol content increased up to a
critical value for each protein. Beyond this, critical value order decreased.115
The result of the deconvolution for wheat gluten is summarized in Figure 7.18.
FT-IR analysis of film blown thermoplastic zein plasticized with polyethylene
glycol highlighted the interdependence of structure and processing. Different
batches of zein powder contained different ratios of b-sheets to a-helices and the
best blown films were prepared from those with largest relative a-helical content.
In turn, processing increased the a-helical content and decreased the presence of
ordered b-sheet regions.103 Cross-links induced by g-radiation in whey, casein
and soya films appeared to have reduced b-sheet regions.116
FT-IR analysis of thermoplastic sheets containing soy protein isolate and
glycerol showed no change in the characteristic peaks of the individual com-
ponents indicating that no covalent interactions formed between them.117 In
contrast, soy protein plastics plasticized with both e-caprolactone and glycerol
did not show peaks characteristic of caprolactone, indicating it had been
consumed.86
Synthesis and Characterization of Thermoplastic Agro-polymers 231
Figure 7.17 FTIR spectrogram of thermoplastic zein protein. The combined peak for
the amide I region is separated as shown into component peaks relating
to different secondary structures.103 M. Oliviero, E. D. Maio and S.
Iannace, Effect of molecular structure on film blowing ability of ther-
moplastic zein. Journal of Applied Polymer Science, 2010, 115(1), 277–
287. Copyright John Wiley and Sons. Reproduced with permission.
Figure 7.18 Secondary structure content of hot pressed wheat gluten films with gly-
cerol as a plasticizer estimated by deconvolution of amide I region in
FTIR spectrum.115
232 Chapter 7
Starch/plasticizer Interactions. Peaks observed in thermoplastic starch
between 992 and 1200 cm1 have been associated with interaction between
starch molecules and plasticizer and can be used to evaluate the efficiency of
different plasticizers.68
Within this region, a peak at 1020 cm1 can be attributed to C–O stretching
in C–O–C bonds for native starch, while peaks at 1081 and 1156 cm1 are
indicative of C–O stretching in C–O–H bonds.118 In thermoplastic starch,
hydrogen bonding between starch and plasticizer causes absorbance to shift to
lower wavenumbers.118,119 Greater reduction in wavenumber is indicative of
stronger plasticizer/starch interactions.68 This shifting is apparent in the FT-IR
spectra for native starch and TPS plasticized with ethanolamine, as shown in
Figure 7.19. The effects of some plasticizers and ageing on these peaks are listed
in Table 7.5.
As seen in Table 7.5, the peak for C–O–C stretching separates into two peaks
as thermoplastic starch (10 wt% 30 pph glycerol) ages. This split peak is also
seen in TPS with 30 pph glycerol and 1 pph citric acid prior to ageing and is
unchanged after 70 days.102
In thermoplastic starch processed with 100 parts hydrous starch (20 % water
content) and 30 pph glycerol and higher concentrations citric acid (10–40 pph),
the peak for C–O stretching in C–O–C bonds was seen at 1024 cm1. Residual
citric acid was removed by washing in deionized water after processing and
before FT-IR. This peak decreased in size relative to the peak at 1149 cm1
(C–O stretching in C–O–H bonds) as citric acid content increased, indicative of
a reduction in molecular mass.70
Other peaks observed in thermoplastic starch provide further information
about interactions between polysaccharide chains, water and plasticizer. A
peak at 1644 cm1 corresponds to water strictly bonded to starch.68 A peak at
3389 cm1 in native cornstarch is ascribed to free, intermolecular and
intramolecular bound hydroxyl groups and decreased to 3325 cm1 in TPS
plasticized with ethanolamine.119 Again, as with the peaks in Table 7.5, this
indicates H-bonding interactions forming with the plasticizer at the expense of
interactions between chains. Other researchers found a similar peak at 3413
cm1 for native starch.69 After processing with high glycerol contents, this peak
shifted to lower wavenumbers and decreased in intensity. As thermoplastic
starch aged (retrograded), this peak shifted back towards that seen in native
starch. After 70 days, the peak had returned to 3413 cm1 for TPS with 30%
glycerol.69 For higher glycerol contents this shifting slowed, suggesting the
glycerol restricted retrogradation.69
A peak at 2931 cm1 representing C–H stretching in CH2 groups did not shift
in TPS plasticized with ethanolamine, indicating the plasticizer did not interact
with these groups.119
7.3.3.7 XRD/XRS
X-ray diffraction (XRD) and X-ray scattering (XRS) are techniques in which a
sample is exposed to X-rays and the resultant scatter pattern is interpreted to
provide information about the spatial arrangement of atoms. Strictly speaking,
X-ray diffraction refers to the patterns of constructive and destructive inter-
ference due to the scattering of rays by crystal planes. Scattering is a more
234 Chapter 7
general term also applicable to amorphous materials which show broad ‘halo’
peaks, rather than sharp peaks caused by diffraction. In practice, similar
instrumentation is used for both techniques and the terms are often used
interchangeably.78 Wide angle X-ray diffraction (WAXD) and wide angle
X-ray scattering (WAXS) (scattering angle 2y451) are most commonly used
for characterization of crystallinity in polymers.78
For thermoplastic starch and proteins, X-ray scattering techniques provide
information about conformational changes induced by processing or ageing as
well as information about the compatibility of plasticizers and cross-linking
agents. Figure 7.20 shows examples of the plots of intensity versus scattering
angle that are obtained as thermoplastic starches age and as plasticizer content
changes for wheat gluten.
The original crystalline order of native starch is destroyed in the production
of thermoplastic starch, but even below the Tg, chains exhibit enough mobility
and re-order over time.93 This re-forming of ordered crystalline regions is called
retrogradation and can be observed using WAXS.68,69,72,74,119 Different plas-
ticizers and plasticizer content affect the rate of retrogradation and the type of
crystallinity that forms. The types of crystallinity that can be identified using
XRD are listed in Table 7.6.
Maize starch plasticized with 30 wt% glycerol displayed a shift from type-A
crystallinity to type-VH and type-VA. Plasticization with 30 wt% of a mixture
Figure 7.20 WAXS of (a) native starch and glycerol plasticized thermoplastic starch
stored for 0, 30, 60 and 90 days respectively (b–e)119 (Reprinted from
Polymer Degradation and Stability, 90(3), M. F. Huang, H. G. Yu and
X. F. Ma, Ethanolamine as a novel plasticiser for thermoplastic starch,
501–507, Copyright (2005), with permission from Elsevier) and wheat
gluten plasticized with different glycerol contents.115 (Reprinted with
permission from A. I. Athamneh, M. Griffin, M. Whaley and J. R.
Barone, Biomacromolecules, 2008, 9, 3181–3187. Copyright 2008
American Chemical Society.)
Synthesis and Characterization of Thermoplastic Agro-polymers 235
68–69,93
Table 7.6 Types of crystallinity in Starch.
Crystallinity type Description
7.4 Conclusions
Thermoplastic agro-polymers are synthesized from starch and proteins. Both
starch and protein experience a barrier to thermoplastic processing due to only
a small difference between glass transition and degradation temperatures. For
dry, unplasticized starch or protein, degradation occurs prior to the formation
of a flowable melt. To overcome this, synthesis requires addition of plasticizers
to reduce the Tg to temperatures which enable processing. For starch, the
crystalline structure of starch granules also needs to be disrupted to allow
processing and this is called gelatinization. Similarly, in proteins, formation of
Synthesis and Characterization of Thermoplastic Agro-polymers 237
thermoplastic melt disrupts higher order structures and this is called
denaturing.
After processing, both starch and protein agro-polymers suffer the same
drawback. Due to their hydophilicity, water is usually an effective plasticizer
and is required for processing. Unfortunately, this hydrophilicity also causes
the mechanical properties of products made from agro-polymers to depend on
moisture content. Exposure to atmospheres of varying relative humidity leads
to undesirable changes in properties. Furthermore, agro-polymers are often
characterized by inferior mechanical properties compared to synthetic ther-
moplastics. These properties also depend on plasticizer content, although
ductility is obtained at the expense of strength.
The most common techniques used for characterization of thermal properties
and structure are TGA, DSC, DMA, FT-IR and XRD. Each of these techni-
ques is used to elucidate specific aspects of agro-polymers.
TGA is used to assess thermal stability of agro-polymers. Starch and proteins
degrade at temperatures around 200–250 1C which is not much higher than their
glass transition temperatures. The Tg of agro-polymers in their native state and
after thermoplastic processing is often determined by DSC and DMA. These
techniques can also be used to determine the temperatures at which gelatiniza-
tion, denaturing and crystallization occur. As with mechanical properties,
thermal properties are dependent on moisture content and care needs to be taken
to restrict or account for moisture loss by evaporation during thermal analysis.
FT-IR and XRD are used to assess the structural changes induced during
thermoplastic processing and the interaction between plasticsers and polymer
chains. For proteins the native secondary, tertiary and quaternary structures
are destroyed by processing, but new secondary structure-like elements may be
induced. Thermoplastic starch is semi-crystalline and tends to retrograde to
higher crystalline content. XRD is extremely useful to determine the degree of
crystallinity in starch.
The structure–property–processing relationship of agro-polymers is highly
interdependent. A considerable research effort has been devoted to firstly obtain
processible thermoplastics. Now, sustained efforts are required to overcome the
water sensitivity and time-dependent properties of these materials.
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CHAPTER 8
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
Degradable Bioelastomers:
Synthesis and Biodegradation
Q. Y. LIU,1 L. Q. ZHANG2 AND R. SHI3
1
Beijing University of Aeronautics and Astronautics, School of Chemistry
and Environment, Xueyuan Road of Haidian District, 100191, Beijing,
People’s Republic of China; 2 Beijing University of Chemical Technology,
College of Materials Science and Engineering, Beisanhuan East Road of
Chaoyang District, 100029, Beijing, People’s Republic of China; 3 Laboratory
of Bone Tissue Engineering of Beijing Research Institute of Traumatology
and Orthopaedics, Beijing 100035, People’s Republic of China
243
View Online
244 Chapter 8
physical cross-linking); (2) certain flexibility and elasticity providing mechanical
stimulation for tissue engineering constructs; (3) appropriate mechanical properties
especially matching with soft tissues of bodies; (4) easily adjustable and designable
biodegradability by tuning the cross-linking density.
Degradable bioelastomers have presented potential applications particularly
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
in the fields of tissue engineering and controlled drug delivery. As the scaffolds
in tissue engineering, degradable bioelastomers are required to support cell-
oriented growth aimed at the generation of replacement tissues, and to degrade
at a suitable degradation rate matching with the generation of new tissues.
Their role of mechanical stimulation in the engineering of elastic soft tissues has
been demonstrated recently to be important.9–11 As the carriers in drug deliv-
ery, degradable bioelastomers can be driven to release drugs by osmotic pres-
sure at a nearly constant rate,12 which is the same as the traditional silicon
rubbers.13 They do not need subsequent surgical removal after being implanted
in bodies, in common with other degradable biomaterials.
Firstly, based on the typical descriptions of biomaterials,14,15 a degradable
bioelastomer is defined as a biodegradable elastomeric biomaterial, either alone
or as a part of a complex system, being used to direct the course of any ther-
apeutic or diagnostic procedure by control of interactions with components of
living systems in human or veterinary medicine. Secondly, in view of the ASTM
definition of ‘elastomer’, a degradable bioelastomer takes on the characteristics
of elastomer materials, whose glass transition temperature (Tg) is lower than
room and body temperature, and which has the ability of resilience to at least
its 1.25-fold original length within 1 minute when it is stretched to its 1.5-fold
original length and then released. Thirdly, as a very successful biomaterial, the
meaning of a degradable bioelastomer also includes bionics, biomimetics and
being bio-inspired. That is to say, a degradable bioelastomer will show similar
molecular structures and physicochemical properties to the replacement tissue
or its surrounding tissue, which will endow it with the biocompatibility, mainly
referring to blood compatibility, tissue compatibility and biomechanical com-
patibility. For example, as well as collagen and elastin, which are cross-linked
polymers that provide elasticity to the natural extracellular matrix (ECM),
degradable bioelastomers should present matched mechanical properties with
the tissues or tissue components as shown in Table 8.1.16–22
Degradable bioelastomers are generally classified as thermoplastic and
thermoset degradable bioelastomers. Thermoplastic degradable bioelastomers
such as segment polyurethanes including biodegradable structures are usually
phase-separated block copolymers cross-linked by physical interaction. The
soft and rubber-like segments (with a low Tg) give them flexibility, while glassy
or crystallizable segments (with a high Tg or melting temperature) provide
strength and stiffness.23,24 Thermoset degradable bioelastomers are often cross-
linked by covalent bonding, which present very stable network structures.25,26
Thermoplastic degradable bioelastomers have the advantage of being easily
fabricated by melt and solvent processing methods; however, their hard regions
with high Tg or having been crystallized often make them biodegrade in a slow
rate and heterogeneous fashion.27,28 Thermoset degradable bioelastomers are
View Online
very hard to be processed to the desired shapes under the help of heat and
solvent again after solidification, but their biodegradation rate is more uniform,
and the remaining dimension is more stable.
246 Chapter 8
8.2.2 Biodegradation
In order to endow bioelastomers with biodegradability in a particular bio-
logical situation, the bioelastomers should be first designed to contain easily
bond-cleavage segments in their molecular structure. The segments, including
ester, anhydride, amide and orthoester units etc. shown in Scheme 8.1, have
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
CH2
O O O O O
-C-O-CH2- -C-O-C- -C-NH-CH2- -CH2-O-C-O-CH2-
R
ester unit anhydride unit amide unit orthoester unit
248 Chapter 8
polyurethane bioelastomers; poly(e-caprolactone) related bioelastomers;
polylactide related bioelastomers; polycarbonate related bioelastomers; poly-
(glycerol sebacate) bioelastomer and its derivatives; citric acid related polyester
bioelastomers; poly(ether ester) bioelastomers; and poly(ester amide) bioelas-
tomers. Their synthesis and biodegradation are specially introduced, accom-
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
O CH3
O C
OCN (CH2)4 NCO OCN (CH2)4 CH NCO
1,4-BDI LDI
CH3 CH2
C CH NCO
CH3 CH
2CH2
C
OCN (CH2)6 NCO CH3 CH2 NCO
1,6-HDI IPDI
O
(1)
O
150 ºC, 7d
+ HO (CH2)4 OH argon
O O
H O (CH2)5 C n O (CH2)4 O C (CH2)5 O mH
(HO-PCL-OH)
80 ºC, 28h
(2) HO-PCL-OH + OCN (CH2)4 NCO argon
O O
OCN (CH2)4 NHC O-PCL-O CHN (CH2)4 NCO
(3) O O
OCN (CH2)4 NHC O-PCL-O CHN (CH2)4 NCO + HO (CH2)4 OH
80 ºC, 72h argon
O O O O
CHN (CH2)4 NHC O-PCL-O CHN (CH2)4 NHC O (CH2)4 O x
Scheme 8.3 Uncatalysed synthesis of the degradable SPU bioelastomers from PCL
polyester diol, 1,4-BDI and BDO.
View Online
250 Chapter 8
initiated with BDO. Catalyst-free synthesis of the SPU bioelastomers not only
weakened the several side reactions which often existed during the catalysed
synthesis, but also improved the materials’ biocompatibility. Tg of the bio-
elastomers ranged from –45.9 to –58.5 1C. Experiments on the bioelastomers as
meniscal replacements in dogs’ knees were conducted,38 which demonstrated
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
that they were able to prevent rupture of the sutures out of the meniscus
scaffolds during the implantation, and they degraded relatively slowly without
swelling during application.
The degradable SPU bioelastomers from PCL polyester diol, 1,4-BDI and
putrescine or lysine ethyl ester chain extenders at the molar ratio of 1:2:1
were synthesized with the catalysts of stannous octoate (Sn(Oct)2) as shown
in Scheme 8.4.39 Incubation in phosphate buffer solutions for 8 weeks
resulted in mass losses above 50% when using lysine ethyl ester as chain
extender, while the mass losses were above 10% when using putrescine as
chain extender. The degradation products were shown to have no toxicity to
human endothelial cells. When they were endowed with an ability to induce
local angiogenesis by controlled release of a basic fibroblast growth factor
(bFGF), the SPU scaffolds degraded slightly slower in comparison with the
ones loaded with bFGF probably because of the lower water absorption in
phosphate buffer solution at 37 1C, as shown in Figure 8.1.40
The degradable SPU bioelastomers from PCL-b-PEO-b-PCL triblock
copolymers as soft segments, 1,4-BDI and peptide Ala-Ala-Lys (AAK) chain
extenders were prepared.41 PCL-b-PEO-b-PCL polyester diols were first syn-
thesized by ring-opening polymerization of e-CL initiated by poly(ethylene
glycol) with Sn(Oct)2 as catalyst at 120 1C for 24 h under nitrogen atmosphere,
then the SPU bioelastomers were prepared according to the reaction
O O
75 ºC, Sn(Oct)2
HO (CH2)5 C C (CH2)5 OH + OCN (CH2)4 NCO
polycaprolactone diol (Mw=2000)
O O O
H NCO
OCN N C O (CH2)5 C C (CH2)5 O C N
O H
prepolymer
O NH
C
O O O
H NH
N C O (CH2)5 C C (CH2)5 O C N
NH O H
C
O
Scheme 8.4 Synthesis of the degradable SPU bioelastomers from PCL polyester diol,
1,4-BDI and putrescine or lysine ethyl ester.
View Online
Figure 8.1 Effect of degradation time on the weight remaining for the SPU and SPU/
bFGF bioelastomer scaffolds in phosphate buffer solutions (pH ¼ 7.4,
37 1C). Adapted from Guan et al.40
50 ºC, 24h
2 HO CH2CH2 NH2 + OCN (CH2)4 NCO
DMF, no catalyst
O
H H
HO C N N
N N C OH
H H
O
252 Chapter 8
O O
O O 125 ºC , 24h
+ + HO (CH2)4 OH
O Sn(Oct)2
O
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
O O O O
O-HCC y O (CH2)5 C x O (CH2)4 O C (CH2)5 O CCH-O y
n x n
CH3 CH3
(HO-PCLA-OH)
Scheme 8.6 Synthesis of the degradable SPU bioelastomers from PCLA polyester
diol, 1,6-HDI and BDO.
O
dibutyltin oxide
HO-CH-CH2-C-OCH2CH3 + HO (CH2)x OH
CH3 CH3CH2OH
x=4, 6, 8 or 10
ethyl(R,S)-3-hydroxybutyrate
O O
H (O-CH-CH2-C)mO (CH2)xO ( C-CH2-CH-O)n H
CH3 CH3
oligo[(R,S)-3-hydroxybutyrate] diol
Figure 8.2 Polarizing light microscopy (PLM) images of the bioelastomer sample
during degradation in phosphate buffer solutions (pH ¼ 7.4, 37 1C) at
different degradation time: (a) hydrolytic degradation for 24 h, (b) enzy-
matic degradation for 12 h and (c) enzymatic degradation for 24 h.
Adapted from Ding et al.47
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254 Chapter 8
8.3.2 Poly(e-caprolactone) Related Bioelastomers
8.3.2.1 Summary
Poly(e-caprolactone) (PCL) is a rubbery semicrystalline polymer at physiolo-
gical temperature, whose Tg is usually lower than –60 1C, being different from
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
O O O
Cl-C (CH2)4 C-Cl + HO CH=CHC-OH
O O O O
HO-CCH=HC O C (CH2)4 C O CH=CHC-OH
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
O
Cl-S-Cl
O O O O
Cl-CCH=HC O C (CH2)4 C O CH=CHC-Cl
HO-PCL-OH
O O O O
CCH=HC O C (CH2)4 C O CH=CHC O-PCL-O
n
UV light irradiation
Scheme 8.8 Synthesis of the photo-cured PCL-related bioelastomers from PCL diols,
adipic acid and 4-hydroxycinnamic acid.
Figure 8.3 Effect of photo-curing time on the weight losses of the PCL-related
bioelastomers from PCL diols (Mw 1250 g mol1) in phosphate buffer
solutions (pH ¼ 7.4, 37 1C) with Ps. cepacia lipase: (a) 10 min, (b) 30 min,
(c) 60 min and (d) 90 min. Adapted from Nagata and Sato51
were first synthesized at 260 1C under nitrogen atmosphere, then they were cast
on an aluminium plate with a 17wt% DMF solution at 80 1C and further post-
polymerized at 280 1C. Tg of the bioelastomers was within –49 to 67 1C. Their
complete degradation in phosphate buffer solutions with Rh. delemar lipase was
View Online
256 Chapter 8
COOH
HOOC-CH2CHCH2-COOH
(tricarballylic acid)
+ HO-PCL-OH
COOH
HOOC-CH2CHCHCH2-COOH
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
COOH
(meso-1,2,3,4-butanetetracarboxylic acid)
COO-PCL
PCL-OOC-CH2CHCH2-COO-PCL
polycondensation
COO-PCL
PCL-OOC-CH2CHCHCH2-COO-PCL
COO-PCL
Scheme 8.9 Synthesis of the thermoset PCL-related bioelastomers from PCL diols
and tricarballylic acid or meso-1,2,3,4-butanetetracarboxylic acid.
O 130 ºC
+ HO (CH2)6 OH
O Sn(Oct)2
O
O O
H O-HCC xO (CH2)6 O CCH-O y H (HO-PLLA-OH)
CH3 CH3
O
O O triethyamine
HO-PLLA-OH +
DMAP, 1,4-dioxane
O O O O
HO-CCH2CH2C O-PLLA-O CCH2CH2C-OH (HOOC-PLLA-COOH)
SOCl2 DMF
O O O O
Cl-CCH2CH2C O-PLLA-O CCH2CH2C-Cl (ClCO-PLLA-COCl)
0 ºC
ClCO-PLLA-COCl + HO-PCL-OH
pyridine
258 Chapter 8
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
Figure 8.5 Mass loss (a) and molecular weight change (b) of the thermoplastic PCL
related bioelastomers during in vitro degradation in phosphate buffer
solutions (pH ¼ 7.4, 37 1C). Adapted from Jeon et al.54
260 Chapter 8
ABA triblock copolymers that contain immiscible segments where A is a
‘hard’, high Tg or semicrystalline polymer and B is a ‘soft’ amorphous, low Tg
polymer can behave as thermoplastic elastomers. Polylactide-containing ABA
triblock copolymers are usually synthesized by the ring-opening polymerization
of lactide initiated by hydroxyl-capped macromolecular monomers such as
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
O
(1) O
HO-CH2CH2-O-CH2CH2-OH + ZnEt2
CH(CH3)2
(diethylene glycol) CH 100 0C
3
(menthide)
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
CH(CH3)2 O O CH(CH3)2
HO CHCH2CH2CHCH2C xO O C-CH2CHCH2CH2CH OH
O y
CH3 CH3
(HO-PM-OH)
(2) 90 ºC
HO-PM-OH + AlEt3 macroinitiator
O
(3) macroinitiator O 90 ºC
+
O
O O
O
H O-HCC nO-PM-O CCH-O mH
CH3 CH3
(PLA-b-PM-b-PLA bioelastomer)
262 Chapter 8
CH3
100 ºC
+ HO (CH2)4 OH catalyst
O
O
O O
H O-CHCH2C xO (CH2)4 O CCH2CH-O y H (HO-PHB-OH)
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
CH3 CH3
O
O 160 ºC
HO-PHB-OH +
catalyst
O
O
O O
H O-HCC nO-PHB-O CCH-O mH (PLA-PHA-PLA bioelastomer)
CH3 CH3
diols reacted with L-lactide using Sn(Oct)2 as catalyst at 160 1C for obtaining
the final bioelastomers. Tg of the bioelastomers ranged from –0.6 to 4.8 1C,
corresponding to the PHB segments. The biodegradation of the bioelastomers
were not reported.
The thermoplastic PLA-related bioelastomers from poly(1,3-trimethylene
carbonate) (poly(TMC)), which was a rubbery and amorphous polymer with
low Tg of about –25 1C, were prepared as shown in Scheme 8.13, being
poly(LA-TMC-LA) copolymers.66 The a,o-hydroxy terminated poly(TMC)
diols were first synthesized by the ring-opening polymerization of 1,3-tri-
methylene carbonate with 1,6-hexanediol as initiator and Sn(Oct)2 as catalyst
for 3 days at 130 1C, and then the LA monomers (D-lactide, L-lactide or D,L-
lactide) were added into the poly(TMC) diols to polymerize for 3 days at 130 1C
with Sn(Oct)2 for obtaining the final bioelastomers. Tg of the bioelastomers
ranged from –19.5 to 8.2 1C. Diethylene glycol instead of 1,6-hexanediol was
first used to synthesize poly(TMC) diols at 140 1C with Sn(Oct)2 as catalyst,
and then the diols were further polymerized with LA monomers in the same
conditions, following chain extension conducted with 1,6-HDI, other poly-
(LA-TMC-LA) bioelastomers were prepared,67 which possessed Tg of –11.1 to
–1.8 1C. In vitro degradation tests demonstrated that the weight loss of the
bioelastomers was 5–7% after 8 week degradation, and their molecular weight
dropped rapidly within a week to about half of original molecular weight, and
then seemed to be balanced as shown in Figure 8.8.
Polyisoprene (PI) polymers with low Tg were used as soft segments for
preparing the thermoplastic PLA related bioelastomers which were synthesized
by reacting a,o-hydroxyl polyisoprene (HO-PI-OH) with lactide using AlEt3 as
catalyst in toluene at 90 1C as shown in Scheme 8.14.68 The bioelastomers
actually were a kind of PLA-PI-PLA copolymer, which could be achieved in
larger quantities and at higher reaction concentrations after using aluminium
View Online
O
O O Sn(Oct)2
HO-(CH2)6-OH +
3 days
(TMC)
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
O O
HO CH2CH2CH2OC xO-(CH2)6-O COCH2CH2CH2 yOH
[HO-poly(TMC)-OH]
O
O Sn(Oct)2
HO-poly(TMC)-OH +
O 3 days
O O
H O-HCC nO-poly(TMC)-O CCH-O H
m
CH3 CH3
(poly(LA-TMC-LA) bioelastomer)
Figure 8.8 Weight remaining and molecular weight change of the poly(LA-TMC-LA)
bioelastomers during in vitro degradation in phosphate buffer solutions
(pH ¼ 7.4, 40 1C). Source: Kim and Lee.67
264 Chapter 8
O 90 ºC, toluene
HO-PI-OH +
(α,ω-hydroxyl O AlEt3 or Al(iOPr)3
polyisoprene)
O
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
O O
H O-HCC nO-PI-O CCH-O mH (PLA-PI-PLA bioelastomer)
CH3 CH3
and biodegradation, and can be applied in biomedical materials, they are called
PC-related bioelastomers which are actually the PC copolymers and sometimes
chemically cross-linked PC polymers.
(1)
H
HO OH
(4) OCH2Ph
H H
HO OH
(2) O
H H H
HO OH
2(CH3CH2)3N, 0 ºC, THF O O
CH3 H (5)CH2OCH2Ph -(CH3CH2O)2CO
(3) HO OH R3
CH3 R1
HO OH H CH2CH3 R2
(1) R1=R2=R3=H, TMC
H CH3 (2) R1=CH3, R2=R3=H, MTMC
(3) R1=H, R2=R3=CH3, 2,3-DMTMC
(4) R1=R2=H, R3=OCH2Ph, BTMC
(5) R1=H, R2=CH2CH3, R3=CH2OCH2Ph, EBTMC
Scheme 8.15 Synthesis of five kinds of six-membered cyclic carbonates from the
corresponding diols.
View Online
266 Chapter 8
diol prepared are shown in Scheme 8.16. The prepolymers were first prepared in
a stream of nitrogen at 260 1C for 20–40 min in PCD1000 system and for 90–180
min in PCD2000 system. Then, the prepolymers were dissolved in DMF and post-
polymerized at 270 1C for 40–80 min to obtain the final products. Tg of the
bioelastomers was in the range –51 to –41 1C. The degradability of the bioe-
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
lastomers were evaluated in phosphate buffer solutions with Rh. Delemar lipase,
and the results demonstrated that the degradation mechanism was enzyme-
catalyzed hydrolysis from the ester linkages between Yt or Y and PCD as well as
the carbonate linkages in the polycarbonate segments. The weight losses of the
bioelastomers increased almost linearly with degradation time, being lower than
10% after 5 day degradation, as shown in Figure 8.9.
COOH
CH2-COOH
CH-COOH
CH2-COOH HOOC COOH
tricarballylic acid (Yt) trimesic acid (Y)
H [O-(CH2)6-OCO]n O-(CH2)-OH
O
polycarbonate diol (PCD)
Scheme 8.16 Molecular structures of tricarballylic acid, trimesic acid and poly-
carbonate diol used in the preparation of thermoset polycarbonate
related bioelastomers.
Figure 8.9 Weight loss of the thermoset PC-related bioelastomers against degrada-
tion time in phosphate buffer solutions (pH ¼ 7.2, 37 1C) with Rh. delemar
lipase. Source: Nagata et al.77
View Online
O
O O 130 ºC
HO-CH2CHCH2-OH +
Sn(Oct)2
OH
(glycerol) (trimethylene carbonate)
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
O O
H O-(CH2)3-O-C x O-CH2CHCH2-O C-O(CH2)3-O y H
O C-O(CH2)3-O z H
O
(hydroxyl group-terminated poly(trimethylene carbonate))
HC-OC 2 5
H
O O
O DMAP, DCC HO-CCH=CHC-OC2H5
CCH=C
O O O
O-(CH2)3-O-C x O-CH2CHCH2-O C-O(CH2)3-O
y O
O C-O(CH2)3-O
CC H
O O
z
CCH
=CH
(ethyl fumarate-functionalized poly(trimethylene carbonate))
O
=CH
C-O 2
UV
O
C H5
C-OC 2
268 Chapter 8
OH
(1) 120 ºC, 24h
HO-CH2CHCH2-OH + HOOC (CH2)8 COOH
(2) 120 ºC, 30 mTorr
OR
OC (CH2)8 COO-CH2CHCH2-O n R=H, or polymer chain
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
Scheme 8.18 Synthesis of the thermoset PGS bioelastomers from glycerol and sebacic
acid.
addition, glycerol and copolymers containing sebacic unit have been approved
for use in medical fields.81 Based on the facts, using glycerol and sebacic acid as
the monomers to prepare degradable bioelastomers is a very good strategy. The
thermoset degradable poly(glycerol sebacate) (PGS) bioelastomers are the most
excellent representative, and have been synthesized by directly melt poly-
condensing glycerol with sebacic acid at the molar ratio of 1/1, as shown in
Scheme 8.18.82 They are being studied for varied potential applications in soft
tissue engineering such as vascular regeneration, myocardial tissue repairing
and retinal tissue engineering.83–89 They have also been researched as drug
carriers.90 Tg of the thermoset PGS bioelastomers were below –80 1C. Their
degradation in vitro was difficult to correlate with the in vivo degradation, and
the mass loss reached 15% after 10 weeks degradation in phosphate buffer
solutions, whereas the complete degradation was observed after 6 weeks in vivo.
Unlike poly(DL-lactide-co-glycolide) which degraded mostly by bulk degra-
dation, in vivo degradation of the PGS bioelastomers was dominated by surface
erosion as indicated by linear mass loss with time, preservation of implant
geometry, better retention of mechanical strength, absence of surface cracks,
and minimal water uptake.91
When designing the thermoset PGS bioelastomers, five hypotheses have been
put forward: (1) endowing them with good mechanical properties by covalent
cross-linking and hydrogen bonding; (2) making them form three-dimensional
networks through the reaction between the trifunctional and difunctional
monomers; (3) controlling their cross-linking density at a low value to make
them very elastic; (4) intensifying their hydrolysis while weakening the enzyme-
catalyzed degradation by inducing ester bonds in molecular chains; (5) lowering
the probability of their heterogeneous degradation by creating cross-linking
bonds which are the same as the ester bonds of the main chains. The derivatives
stand for the bioelastomers which are designed and prepared because of the
inspiration from the above hypotheses and the PGS bioelastomers.
Figure 8.10 Weight loss–degradation time curves of the TM-PGS bioelastomers pre-
pared by two steps using the PGS prepolymers with different molecular
weights (phosphate buffer solutions, pH ¼ 7.4, 37 1C): (a) Mn ¼ 1681 g
mol–1, (b) Mn ¼ 2426 g mol–1, (c) 4429 g mol–1. Source: Liu et al.94
270 Chapter 8
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
Figure 8.11 Mass loss of the Acr-PGS bioelastomers during the in vivo (black, dorsal
pocket of male Sprague-Dawley rats) and in vitro (white, phosphate
buffer solutions, pH ¼ 7.2, 37 1C) degradation at 2, 4 and 8 weeks.
Source: Ifkovits et al.96
OH OR O O
HO-CH2CHCHCHCH2-OH O-CH2CHCHCHCH2-O-C-(CH2)8-C n
OH OH OR OR
(xylitol) poly(xylitol sebacate)
OH OR O O
HO-CH2CHCHCHCHCH2-OH O-CH2CHCHCHCHCH2-O-C-(CH2)8-C n
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
OH OH OH OR OROR
(sorbitol) poly(sorbitol sebacate)
HO OH RO OR O O
HO-CH2CHCHCHCHCH2-OH O-CH2CHCHCHCHCH2-O-C-(CH2)8-C n
OH OH OROR
(mannitol) poly(mannitol sebacate)
HO OH RO OR
HO O O O O O
OH OR O O
HO OH OH RO OR O-C-(CH2)8-C n
OH OH OR OR
(maltitol) poly(maltitol sebacate)
R=H, or polymer chain
Scheme 8.19 Molecular structures of the polyol monomers and corresponding PPS
bioelastomers.
272 Chapter 8
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
Figure 8.12 Weight loss of the POC bioelastomers prepared under different
conditions during degradation in phosphate buffer solutions (pH ¼ 7.4,
37 1C). Source: Yang et al.100
OH
polycondensation
HOOC-CH2CCH2-COOH + HO-(CH2)x-OH
COOH
aliphatic diol
citric acid (x=6,8,10 or 12)
OR
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
Scheme 8.20 Synthesis of poly(diol citrate) bioelastomers from citric acid and varied
aliphatic diols.
274 Chapter 8
COOH
HOOC COOH + HO-(CH2)8-OH
OH
HO OH
O O
OH O
O
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
O O
+ (glycerol 1,3-diglycerolate diacrylate)
polycondensation
O
HO O
O OH
O
(bis(hydroxypropylfumarate))
COOR COOR
OC CO O-(CH2)8-O x OC CO O O
O y
OR OR O
(A) OR O
O
O O
COOR COOR
O
OC CO O-(CH2)8-O x OC CO O O
OR OR O O y
(B) O
R=H, or polymer chain
Scheme 8.21 Synthesis of the citric acid based unsaturated prepolymers from citric
acid 1,8-octanediol and glycerol 1,3-diglycerolate diacrylate or bis(hy-
droxypropylfumarate): (A) acrylated prepolymers; (B) fumarate-con-
taining prepolymers.
Figure 8.13 Weight loss of the PEC bioelastomers during degradation in phosphate
buffer solutions (pH ¼ 7.4, 37 1C) at the molar ratio of 10/9 (PEO/citric
acid) with different post-polymerizing time. Source: Ding et al.109
View Online
OH
130 ºC, 1kPa
HO-CH2CHCH2-OH + HOOC (CH2)8 COOH
OR
HOOC (CH2)8 COO-CH2CHCH2 n OH
OR OH
HOOC (CH2)8 COO-CH2CHCH2 n OH + HOOC-CH2CCH2-COOH
OH COOH
120 ºC
RO OC-CH2CCH2-CO OR R=H, or polymer chain
m
COOR
Scheme 8.22 Synthesis of the PGSC bioelastomers from glycerol, sebacic acid and
citric acid.
View Online
276 Chapter 8
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
Figure 8.14 Mass loss–degradation time curves of the PGSC bioelastomers with
different thermal-curing times at molar ratios of 4/4/1 and 4/4/0.6 in
phosphate buffer solutions (pH ¼ 7.4, 37 1C). Source: Liu et al.112
The POCS bioelastomers including more sebacic units in the molecular chains
degraded more slowly in phosphate buffer solutions, and the mass losses could
reach 64.1% after 6 h.
The urethane-doped POC bioelastomers were prepared as follows:116 in
the first step, citric acid and 1,8-octanediol were bulk polymerized at 140 1C at
the molar ratio of 1:1.1 for achieving POC prepolymers; in the second step, the
POC prepolymers were dissolved in 1,4-dioxane to form a 3% (wt/wt) solution,
and reacted with HDI with stannous octoate as catalyst (0.1%wt) at 55 1C for
preparing urethane-doped POC prepolymers at different feeding ratios of 1:0.6,
1:0.9 and 1:1.2 (POC prepolymer/HDI); in the third step, the urethane-doped
POC prepolymers were cast into a Teflon mould and allowed to dry with a
laminar airflow until all the solvents were evaporated, and then were further
maintained in an oven at 80 1C to obtain the cured urethane-doped POC
bioelastomers. Tg of the bioelastomers ranged from 0.64 to 5.20 1C. The
urethane-doped POC bioelastomers with higher isocyanate content exhibited
faster degradation rates, and their mass losses were lower than 16% after 2
months in phosphate buffer solutions.
of soft segments.
The thermoplastic poly(ethylene glycol)/poly(butylene terephthalate) (PEG/
PBT) is the most representative and mature poly(ether ester) bioelastomer,
whose molecular structure is shown in Scheme 8.23, being usually synthesized
by ester exchange methods.117–119 Tg of the PEG/PBT bioelastomers is lower
than 30 1C. Their in vitro and in vivo degradation happens both by hydrolysis
and oxidation, in which hydrolysis is the main degradation mechanism.120,121
The bioelastomers do not induce any adverse affects on the surrounding tissues
and show a satisfactory biocompatibility. The PEG/PBT bioelastomers have
been studied for use as skin substitutes,122 elastomeric bioactive coatings on
load-bearing dental and hip implants,123 adhesion barriers,124 bone replace-
ments125 and lysozyme delivery carriers.126 When the phosphate groups are
introduced into their molecular chains, another poly(ether ester) bioelastomer
similar to the PEG/PBT bioelastomers are achieved, whose molecular structure
is shown in Scheme 8.24, having been studied as nerve guide conduit materi-
als127 and drug delivery carriers.128
By bulk polymerization with a two-step melt polycondensation, a type of
aliphatic poly(ether ester) bioelastomer called poly(butylene succinate)-co-
poly(propylene glycol) (PBS-co-PPG) were prepared.129 In the first step, suc-
cinic acid reacted with 1,4-butanediol at 180 1C until the theoretical amount of
water was removed for achieving the desired PBS; in the second step, poly-
(propylene glycol) (PPG) was added into the reaction system, and the poly-
condensation was carried out at 220–230 1C with titanium butoxide as catalyst
under the pressure of 10–15 Pa for obtaining the final PBS-co-PPG bioelas-
tomers. The molecular structure of the poly(ether ester) bioelastomers is shown
in Scheme 8.25. T g of the bioelastomers ranged from –60.7 to –46.6 1C. As
shown in Figure 8.15, the weight losses of the bioelastomers could reach 33%
O O O O
(OCH2CH2)nO-C C x O-(CH2)4-O-C Cy
m
O O O O O
(CH2)2-O-C C-O-(CH2)2-O-P m O-(CH2)2-O-C C-O
2n
OCH2CH3
278 Chapter 8
O O O O
C(CH2)2CO(CH2)4O C(CH2)2COCH2CH (OCH2CH)n O y
x
CH3 CH3
Figure 8.15 Weight loss of the PBS-co-PPG bioelastomers and PBS during degra-
dation in phosphate buffer solutions (pH ¼ 6.86, 45 1C): in the abbre-
viation of PBSxPy, ‘‘x’’, ‘‘P’’ and ‘‘y’’ separately represent the mass
fraction of PBS such as 90%, PPG soft segment and molecular weight
of PPG (1 stands for 1000 g mol–1, and 2 stands for 2000 g mol–1).
Source: Huang et al.129
HO-CH2-CH-CH2-OH
H2N-CH2-CH-CH2-NH2 OH
OH + (Glycerol)
or
(1,3-diamino-2-hydroxy-propane) OH
HO-CH2-CH-CH-CH2-OH
OH
(D,L-threitol)
O O O O
C (CH2)8 CO N-CH2-CH-CH2-N C (CH2)8 CO CH2-CH-CH2-O
OR2 n m
R1 R1 OR2
or
O O O O OR2
C (CH2)8 CO N-CH2-CH-CH2-N C (CH2)8 CO CH2-CH-CH-CH2-O
OR2 n m
R1 R1 OR2
R1=polyamide chain, R2=polyester chain
280 Chapter 8
separately exhibited 97.0% and 44.3% mass losses after 6 week in vitro
degradation (sodium acetate buffer solution, pH ¼ 5.2, 37 1C), while the
poly(DAHP-T-SA) bioelastomers cured for 24 h and 48 h lost 70.4% and
42.8% of mass, respectively. The in vivo degradation half-lives of the bioelas-
tomers were up to 20 months.
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
(A)
O
C-O-CH2CH2-O-C-C8H16-CH=CH
O
O-C-H16C8
O
(B)
C-O-C9H18-CH=CH
O
O-C-H16C8
O
Scheme 8.27 Molecular structures of two kinds of cyclic bile acids: (A) 38-membered
ring, (B) 35-membered ring.
View Online
(A)
O
C-O-CH2CH2-O-C-C8H16-CH=CH
O n
O-C-H16C8
O
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
(B)
C-O-C9H18-CH=CH
O m
O-C-H16C8
O
Scheme 8.28 Molecular structures of two kinds of bile acid-based bioelastomers: (A)
from 38-membered cyclic bile acid, (B) from 35-membered cyclic bile acid.
COOH
140 ºC
HOOC-CH2CHCH2-COOH + HO-(CH2)n-OH
stannous 2-ethylhexanoate
(tricarballylic acid) (alkylene diols, n=6, 8, 10, 12)
COO -(CH2)n-OH
H O-(CH2)n-OOC-CH2CHCH2-COO -(CH2)n-O H
m
(poly(diol-tricarballylate) prepolymer)
COO -(CH2)n-OH O
H O-(CH2)n-OOC-CH2CHCH2-COO -(CH2)n-O H + CH2=CH-C-Cl
m
(acryloylchloride)
O O
CH2=CH-C O-(CH2)n-OOC-CH2CHCH2-COO -(CH2)n-O C-CH2=CH
m
COO -(CH2)n-O- C-CH2=CH
(acrylated poly(diol-tricarballylate)) O
282 Chapter 8
diol-co-tricarballylate) (PDDT), were first synthesized at 140 1C with stannous
2-ethylhexanoate as catalyst under vacuum (50.8 mmHg). Then, acrylation of
the PDT prepolymers was carried out by reacting acryloyl chloride with the
terminal hydroxyl groups of the prepolymers in the presence of triethylamine
and 4-dimethylaminopyridine in an ice bath. Finally, the acrylated poly(diol-
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
COOH O
O
HO-(CH2)8-OH + HOOC-CH2CCH2-COOH + O
(1,8-octanediol) OH
(citric acid) (maleic anhydride)
140 ºC 3h
COOR
O-(CH2)8- OOC-CH2CCH2-COO -(CH2)8-OOC-CH=CH-COO n
OR R=H or polymer chain
[poly(octamethylene maleate (anhydride) citrate) prepolymer]
O OH
HOOC-C-(CH2)2-COOH + HO-CH2-CH-(CH2)n-CH2-OH
(α-ketoglutaric acid) (triols, n=0, 1, 3)
O OR
polycondensation
OC-C-(CH2)2-COO-CH2-CH-(CH2)n-CH2-O m
(poly(triol α-ketoglutarate) bioelastomer)
Figure 8.16 Mass loss of the poly(triol a-ketoglutarate) bioelastomers such as PGa,
PBa and PHa during degradation in phosphate buffer solutions (pH ¼
7.4, 37 1C) under the conditions of different curing temperature and time.
Adapted from Barrett and Yousaf.146
View Online
284 Chapter 8
8.4 Conclusions
It can be seen that degradable bioelastomers such as degradable SPU bioe-
lastomers, PCL-related bioelastomers, PLA-related bioelastomers, PC-related
bioelastomers, PGS bioelastomer and its derivatives, citric acid-related poly-
ester bioelastomers, poly(ether ester) bioelastomers, PEA bioelastomers and so
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
Acknowledgements
This work was supported by Project of National Science Foundation for
Young Scientists of China (Grant No. 51003003) and Key Project of Natural
Science Foundation of China (Grant No. 50933001).
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Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00243
Functionalization of
Poly(L-lactide) and
Applications of the
Functionalized Poly(L-lactide)
XIULI HU AND XIABIN JING
9.1 Introduction
The term ‘biodegradable polymer’ refers to a class of polymer materials that
can be degraded or hydrolysed in vivo into small molecules, which can be
absorbed through metabolism or excreted by the body, or destroyed by
microorganisms in the environment. With the increasing severity of environ-
mental problems and awareness of environmental protection, the disposal
problem of the non-degradable petroleum-based plastics has raised the demand
for biodegradable polymers as means of reducing the environmental impact.
Biodegradable materials and environmentally friendly products are becoming
one of research hotspots nowadays.
Among the biodegradable polymers under investigation, poly(lactic acid)
(PLA) has received the most attention. Its prominent role is based on the
following reasons: (1) its raw material, L-lactic acid, can be efficiently produced
by fermentation from renewable resources such as starch and other
291
View Online
292 Chapter 9
polysaccharides, which are easily available from corn, sugar beet, sugar cane,
potatoes and other biomasses; (2) PLA is biodegradable and biocompatible and
has represented its potential applications in a number of growing technologies
such as drug delivery, sutures, orthopaedics and temporary matrixes or tissue
engineering scaffolds; (3) PLA has high strength and has excellent shaping and
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00291
Cracking O O
O and
Dehydration cyclization Catalyst HO H
HO O
OH Oligomer O
O n
CH3 CH3
O
Over the past few decades, many functionalized monomers for chemical
modification of PLA were designed and synthesized, including (1) morpholine-
2,5-dione derivatives, (2) a-amino acid N-carboxyanhydrides (NCA), (3) cyclic
carbonates, and (4) lactones. The monomer units are introduced into the PLA
backbones via ROP with lactide and the obtained copolymers are usually
random copolymers, block copolymers or block copolymers with a third
hydrophilic block – poly(ethylene glycol) (PEG).
O
H
BrCHR 2COBr DMF/TEA O
NH 2CHCOOH BrCHR 2CONHCHCOOH R1
p-Dioxane/TEA H NH
R1 R1 R2
O
O O
H O R1 O
O R1 O Catalyst
+ O O
H NH O * N *
R2 m H n
O O R2
O
294 Chapter 9
Table 9.1 Structure of morpholine-2,5-dione derivatives and polymers
therefrom.
Polymer after
Entry Monomer (a) Polymer (b) deprotection (c) Ref.
O
H O R2 O R2
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00291
H H
O R1 N N
NH * O * O
H n n
R2 R1 O R1' O
O
O O
NH2 O R
THF
+ CCl 3OCOCCl 3 O
R OH N
H O
296 Chapter 9
Table 9.2 Structures of a-amino acid NCA derivatives.
Polymer after
Entry Monomer (a) Polymer (b) deprotection Ref.
O O
O H H
R N N
O
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00291
HN n
O R R'
catalyst. The pendant amine groups can be used to attach drug and protein
molecules. Disadvantages connected with these polypeptides are the inevitable
use of protecting group chemistry during monomer synthesis. The most
often used a-amino-protecting groups are the 9-fluorenylmethoxycarbonyl
(Fmoc),32,33 the benzyloxycarbonyl (Z) and the tert-butyloxycarbonyl (Boc)
groups.34 The protecting groups should confer solubility in the most common
solvents and prevent or minimize epimerization during the coupling, and their
removal should be fast, efficient and free of side reactions and should render
easily eliminatable by-products. Albericio et al.35 have recently written an
excellent review on the protecting groups of amino acids. NCA monomers
based on DL-allylglycine36 and g-propargyl L-glutamate37 were synthesized
and post-functionalization was carried out via thiol-ene chemistry and azide-
alkyne click chemistry, in which protection and deprotection processes were
not needed. Besides linear polypeptides, other architectures such as cyclic,
star shaped and brushed polypeptides have been extensively investigated.
Hadjichristidis22 described these structures in a related review. These poly-
peptides possess the ability to form a-helix and b-sheet motifs. These secondary
structures contribute significantly to the self-assembling character of polypeptide
chains, leading to novel supramolecular structure with potential biomedical
and pharmaceutical applications. For example, Sun26 reported the direct for-
mation of giant vesicles from poly(L-lysine)-block-poly(L-phenylalanine)
(PLL-b-PPA) block copolymers in water solution. Deming et al.38,39 synthe-
sized a series of diblock copolypeptides via a living polymerization of several
NCAs. These copolypeptides were used in the synthesis of ordered silica
structures and inorganic hollow spheres. Polypeptide and its copolymers have
shown good mechanical properties, film forming and spinnability and they
have great potential use in tissue engineering scaffolds. In comparison to
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O
OH OH O
TEA O O
+ CH2CH3
Cl O
R1 R2 THF
R1 R2
298
1 R1 ¼ H R1 ¼ H R1’ ¼ H 61, 62
R2 ¼ OCH2C6H5 R2 ¼ OCH2C6H5 R2’ ¼ OH
2 R1, R2 ¼ (CH2O)2CHC6H5 R1, R2 ¼ (CH2O)2CHC6H5 R1’, R2’ ¼ (CH2OH)2 51
3 R1, R2 ¼ (CH2O)2C(CH3)2 R1, R2 ¼ (CH2O)2C(CH3)2 R1’, R2’ ¼ (CH2OH)2 66
4 R1 ¼ CH3 R1 ¼ CH3 R1’ ¼ CH3 70–72, 73
R2 ¼ COOCH2C6H5 R2 ¼ COOCH2C6H5 R2’ ¼ COOH
5 R1 ¼ H R1 ¼ H R1’ ¼ H 46, 53
R2 ¼ NHCOOCH2C6H5 R2 ¼ NHCOOCH2C6H5 R2’ ¼ NH2
6 R1 ¼ CH3 R1 ¼ CH3 67
R2 ¼ CH2OOCCH ¼ CHC6H5 R2 ¼ CH2OOCCH ¼ CHC6H5
7 R1 ¼ CH3 R1 ¼ CH3 68
R2 ¼ COOCH2CH ¼ CH2 R2 ¼ COOCH2CH ¼ CH2
8 R1 ¼ CH3 R1 ¼ CH3 69
R2 ¼ COOCH2CCH R2 ¼ COOCH2CCH
Chapter 9
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9.2.4 Lactones
Functionalized aliphatic polyesters based on lactones have been extensively
investigated. The synthesized functionalized lactones include four-membered,
six-membered and seven-membered lactones, and among them, substituted
caprolactone (CL) is the most commonly used. Lou4 has given a detailed review
on these functionalized lactones and so we will not discuss them here.
300 Chapter 9
hydrophilicity. By attaching a number of biologically active compounds, such
as drugs, peptides, sugars and other substances, these polyesters were endowed
with specific biological activities, making them applicable to separation,
diagnosis, treatment and other functions. With the development of modern bio-
medicine, these intelligent polymers are in higher demand. In the following, the
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302 Chapter 9
groups. The anticancer drug adriamycin was conjugated to glutamic acid units
through an amide bond. Their results suggested that conjugation of a receptor-
homing ligand to the PEG-terminal of the polymer-drug conjugate enhanced the
targeted delivery of anticancer agents. Zhu90 conjugated the anthracyclin
antineoplastic agent doxorubicin (adriamycin) to DalB02, an IgG1 kappa
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is free in plasma, it dissociates into ab dimmers, which are rapidly filtered out in
the kidney. The ‘first-generation’ haemoglobin-products are based on the
observations that cross-linking Hb can overcome subunit dissociation and
renal toxicity,103 including polymerized Hbs, intramolecularly cross-linked Hbs
and perfluorocarbon emulsions. The ‘second-generation’ products are based on
a better understanding of the mechanisms of vasoconstriction.104 But clinical
trials have been disappointing because of unexpected toxicity. From the early
1990s to the present, researchers have been dedicated to the development of the
third generation of red blood cell substitutes. Haemoglobin vesicles (HbV)
prepared by encapsulating Hb molecules into lipid or polymeric vesicles105,106
exhibited much better performance than chemically modified Hbs. The HbVs,
with a diameter of 100–250 nm, have a layer of membrane which prevents the
Hb molecules from contacting directly with the immunological system107 or
penetrating vascular wall.108 It is expected to be the most promising artificial
oxygen carrier. The matrix used for HbVs should combine good biocompat-
ibility, non-toxicity and non-immunogenicity. In 1957, Chang109 first prepared
nylon and colloidin microcapsules. Many studies were reported on liposome-
encapsulated Hb (LEH) since Djordjevich and Miller established the method in
1977. Recently, biodegradable polymer PLA was more and more studied as the
HbV matrix due to its favourable properties such as: (1) its biocompatibility
and biodegradability, (2) its chemical structure diversity by copolymerization
with other monomers, and (3) good mechanical properties compared with
traditional HbV matrixes. Chang’s group first tried to prepare artificial blood
cells with PLA materials in 1976.110 Recently, they reported some approaches
to nanoscale Hb encapsulation with PEG-PLA.107,111 Rameez et al.105
described polymersome-encapsulated haemoglobin (PEH). The polymersome
was self-assembled from biodegradable and biocompatible amphiphilic diblock
copolymers composed of poly(ethylene oxide) (PEO), poly(caprolactone)
(PCL) and PLA and their results indicated that Hb encapsulation did not affect
the oxygen binding properties of Hb. Sun et al.106 constructed an oxygen carrier
by encapsulating carbonylated haemoglobin (CO-Hb) molecules into poly-
peptide vesicles made from poly(L-lysine)-block-poly(L-phenylalanine) (PLL-
b-PPA) diblock copolymers in aqueous medium. The CO-Hb encapsulated in
the PLL-b-PPA vesicles was more stable than free CO-Hb under ambient
conditions. In the presence of an O2 atmosphere, the CO-Hb in the vesicle
could be converted into oxygen-binding haemoglobin (O2-Hb) under irradia-
tion of visible light. In another paper,69 azidized haemoglobin molecules were
conjugated onto polymer micelles containing propargyl groups via click reac-
tion between the propargyl groups in the micelles and the azido groups in the
haemoglobin molecules. The propargyl groups were attached to the core
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304 Chapter 9
surface of the micelles and expected to react with the azido groups without
difficulty and the haemoglobin molecules were hidden in the PEG corona of the
micelles, being protected against the immunological systems. Results indicated
that the conjugated haemoglobins retained their O2-binding ability.
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9.4 Conclusions
PLA and its copolymers offer prospective applications in a number of fields,
such as tissue engineering scaffolding, drug delivery systems, implant materials,
gene delivery, protein separation and purification and disease diagnosis. We
have reviewed concisely the methods of PLA functionalization and applications
of the functionalized PLAs in drug delivery, artificial oxygen carriers and
protein separation and purification that our group has worked on in the past
decade. More research is needed to get tailored properties with respect to
degradability and strength for specific applications. Moreover, there is a great
potential to use PLA in a number of unexplored applications by replacing the
conventional polymers, where it can contribute a significant role in the form of
composites, copolymers and blends to obtain the required properties for
different applications.
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306 Chapter 9
4. X. D. Lou, C. Detrembleur and R. Jerome, Macromol. Rapid. Commun.,
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33. L. A. Carpino and G. Y. Han, J. Am. Chem. Soc., 1970, 92, 5748.
34. E. Hlebowicz, A. J. Andersen, L. Andersson and B. A. Moss, J. Pept.
Res., 2005, 65, 90.
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Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00291
Biodegradation of Poly
(3-hydroxyalkanoates)
RACHANA BHATT,1, 2 KAMLESH PATEL1 AND
UJJVAL TRIVEDI1
1
BRD School of Biosciences, Sardar Patel University, Vallabh Vidyanagar –
388 120, Gujarat, India; 2 Department of Microbiology, Shri A. N. Patel
Postgraduate Institute, Charotar Education Society, Anand – 388 001,
Gujarat, India
10.1 Introduction
In the second half of twentieth century there evolved a group of materials never
seen before on this earth, plastics – a synthetic product whose production
skyrocketed as soon as it was invented. They have low cost of production, ease
of processing and outstanding mechanical as well as physical properties. They
have heterogeneous properties with many practical uses and all of them possess
a common property of high resistance to natural degradation processes. As a
result, they have replaced wood, cotton, paper and other natural materials in
many applications. They are widely used in packaging, building materials and
commodity as well as hygienic products. The fact that plastics are mostly non-
degradable is the primary cause of increasing amounts of solid waste, whose
major content is synthetic polymers. This is the primary driving force for sci-
entists trying to find a solution to the waste problem. Efforts have been made to
design and develop biodegradable alternatives, sometimes also referred to as
‘bioplastics’. The term bioplastics may refer to biodegradable plastics and/or
plastics from a biological origin.
311
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312 Chapter 10
Many living systems (mainly plants and microorganisms) produce materials
with great potential for replacing synthetic plastics. One of the typical groups of
materials found in nature is polyhydroxyalkanoates (PHAs). These are natural
polyesters that are produced by many microorganisms, especially bacteria, as a
means to store carbon and energy. PHAs are synthesized and deposited in the
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00311
Structural proteins
PHA depolymerase
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PHA synthase
Phospholipid monolayer
Regulatory proteins
Amorphous PHA
Hydroxyacid
(Monomer)
Poly(hydroxyacid)
Figure 10.3 Chemical structure of PHAs. Side chain ¼ CH3 to C2H5 are short chain
length PHAs. Side chain ¼ C3H7 to C11H23 are medium chain length
PHAs. Side chain4C14H29 are long chain length PHAs.
314 Chapter 10
synthetic polymer. Contrary to PP, polyhydroxybutyrate is biodegradable in a
reasonable time period. PHB is degraded under usual waste disposal conditions
in, roughly estimated, several months. However, for polypropylene, we have to
wait tens of years or centuries!7
Thus, owing to the thermoplastic properties of PHAs and their biodegrad-
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appropriate microorganisms
environmental conditions conducive for the growth of microorganisms
vulnerability of the plastic to microbial attack.
316 Chapter 10
10.3 Biodegradation of Polyhydroxyalkanoates
As described earlier, PHAs are accumulated in the form of granules and serve as
storage compounds for carbon and energy under growth-limiting conditions.22
Thus, PHA can be degraded within the cell for its own growth and sustainability,
i.e. intracellular degradation of PHA. Intracellular degradation is the active
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318 Chapter 10
Table 10.2 PHA-degrading bacteria reported for their ability to degrade
different PHAs.
Group Polymer degraded Representative strains References
Aspergillus spp.
A. penicilloides PHB 38
A. fumigatus PHB, poly(3HB-co-3HV) 39, 40
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A. fumigatus PHB 41
A. fumigatus Pdf1 PHB, PHV, poly(3HB-co-3HV) 42
A. fumigatus 202 PHB 43
Penicillium spp.
P. funiculosum PHB 44
P. adametzii PHB 38
P. daleae PHB 38
P. chermisinum PHB 45
P. pinophilium PHB 46
P. funiculosum PHB 47
P. funiculosum LAR 18 PHB 48
P. minioluteum LAR 14 PHB 48
P. simplicissimum LAR13 PHB 49
Penicillium sp. DS9713a-01 PHB 50
Penicillium sp. DS9701-09a PHB 51
Paecilomyces spp.
Paecilomyces marquandii PHB 38
Paecilomyces lilacinus D218 PHB 52
Paecilomyces lilacinus F4-5 PHB, poly (3HB-co-3HV) 53
Other species
Candida guilliermondii PHB 54
Cephalosporium sp. PHB 55
Cladosporium sp. PHB 55
Debaromyces hansenii PHB 54
Emericellopsis minima W2 PHB, poly (3HB-co-3HV) 56
Eupenicillium sp. IMI 300465 PHB 57
Mucor sp. PHB 55
320 Chapter 10
Extracellular bacterial PHA depolymerases are non-glycosylated in nature,
in contrast to all fungal PHA depolymerases which possess additional carbo-
hydrate moieties at their N-terminal. Data indicates that this carbohydrate
moiety becomes covalently attached to the depolymerase during secretion of
the enzyme across the cell wall and the presence of carbohydrate is not essential
for the activity of the enzyme.59 It is possible that glycosylation enhances the
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N-terminal C-terminal
SP Cataltic Domain Linker SBD
of enzyme of enzyme
322 Chapter 10
(1) The signal peptide (SP) is 22–58 amino acids long. It is responsible for
transport of the enzyme across the cytoplasmic membrane of the cell.
During the secretion process of the enzyme this signal peptide is cleaved
off by signal peptidases.
(2) A large catalytic domain (320–400 amino acids) is situated at the N
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00311
These three functional domains (namely catalytic domain, linker domain and
substrate-binding domain) are essential for the enzymatic degradation of water-
insoluble polymers. Hence it has been proposed that the degradation of the
polymer should proceed in three steps: (1) adsorption of the enzyme to the polymer,
(2) non-hydrolytic disruption of the structure of the polymer, and (3) hydrolysis.84
during secretion of the enzyme across the cell wall. The presence of carbohydrate
is not essential for activity of P. lemoignei depolymerases because recombinant
E. coli or B. subtilis strains harbouring the corresponding depolymerase gene
synthesized the non-glycosylated form of the enzyme but had almost the same
specific activity. It is possible that glycosylation enhances the resistance of the
exoenzyme to elevated temperature and/or to hydrolytic cleavage by proteases of
competing microorganisms. PHB depolymerases of P. lemoignei apparently
catalyse the reverse reaction, i.e. the synthesis of esters for transesterification, if
the reaction is performed in the absence of water (e.g. in solvents).62,63
(1) The oxygen atom of Ser39 makes a nucleophilic attack on the car-
bonyl carbon atom of a bound PHB chain. The His155-Asp121
hydrogen bonding system may enhance the nucleophilicity of the
hydroxyl group of Ser39.
(2) Oxygen atom of Ser39 binds to the carbonyl carbon of PHB making a
tetrahedral intermediate having a formal negative charge on the oxy-
gen atom derived from the carbonyl group. This charge is stabilized by
interactions with the –NH group from amino acids in a site termed as
the oxyanion hole. These interactions also help to stabilize the tran-
sition state that precedes the formation of tetrahedral intermediate.
(3) A proton transfers from the positively charged His155 to the O
formed by the cleavage of the ester bond (acyl-enzyme intermediate).
(4) The first product is now free to depart from the enzyme, completing
the first stage of hydrolytic reaction – acylation of the enzyme.
(5) The next stage is deacylation of enzyme and it begins with the entry
of a water molecule. The ester group of the acyl-enzyme is now
hydrolysed by a process that essentially repeats steps 2–4.
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324 Chapter 10
a
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Active Site
Oxyanion hole
b
PHB
Oxyanion hole
Tetrahedral
Intermediate
Figure 10.6 A proposed mechanism for the action of the PHB depolymerase from
P. funiculosum70 (Image modified). a: catalytic triad in active site of PHB
depolymerase; b: catalytic mechanism.
(6) His155 draws a proton away from water. The resulting OH ion
attacks carbonyl carbon atom of the acyl group, forming a tetra-
hedral intermediate.
(7) This structure breaks down to release the carboxylic acid product.
(8) Finally, the carboxylic acid product is released and the enzyme can
enter into another round of catalysis.
definitely affect the degradation rates of the polymers. The more com-
plex the polymeric composition, the more time it will take to degrade.
326 Chapter 10
acetyl-CoA via acetoacetyl-CoA and 3HB-CoA to PHB by b-ketothiolase,
acetoacetyl CoA reductase and PHB synthase, respectively (PHB biosynthesis
sequence) and from PHB via 3HB and acetoacetate to acetoacetyl-CoA and
acetyl-CoA by intracellular PHB (i-PHB) depolymerase, 3HB dehydrogenase,
acetocaetate:succinyl-CoA transferase and ketothiolase (PHB degradation
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sequence). However, the recent research efforts have led to a possibility that
thiolysis of PHB would be a more appropriate metabolic route to be followed
by the organism to degrade PHB in order to make the whole process energy
efficient. But the major role for the thiolysis of PHB will be played by intra-
cellular PHB depolymerase, leading to a degradation sequence of PHB to
3-hydroxybutryl CoA to acetoacetyl-CoA and acetyl-CoA. It has been reported
that the intracellular PHB degradation due to PHB depolymerase led
to synthesis of 3-hydroxybutyryl-CoA instead of 3-hydroxybutyric acid.110
Thus the mode of action of intracellular PHB depolymerase on PHB is
definitely different from that of extracellular PHB on crystalline PHB. Much of
the details on the mechanism of action of intracellular PHB depolymerase is
yet to be explored.
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Cramm, T. Eitinger, C. Ewering, M. Potter, E. Schwartz, A. Strittmatter,
I. Voss, G. Gottschalk, A. Steinbuchel, B. Friedrich and B. Bowien, Nat.
Biotechnol., 2006, 24, 1257.
114. H. Saegusa, M. Shiraki, C. Kanai and T. Saito, J. Bacteriol., 2001, 183, 94.
115. L. J. Foster, R. W. Lenz and R. C. Fuller, FEMS Microbiol. Lett., 1994,
118, 279.
116. L. J. Foster, E. S. Stuart, A. Tehrani, R. W. Lenz and R. C. Fuller, Int. J.
Biol. Macromol., 1996, 19, 177.
117. L. J. Foster, R. W. Lenz and R. C. Fuller, Int. J. Biol. Macromol., 1999,
26, 187.
118. L. I. de Eugenio, P. Garcia, J. M. Luengo, J. M. Sanz, J. S. Roman, J. L.
Garcia and M. A. Prieto, J. Biol. Chem., 2007, 282, 4951.
CHAPTER 11
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Degradation of Biodegradable
and Green Polymers in the
Composting Environment
ACKMEZ MUDHOO,1 ROMEELA MOHEE,1
GEETA D. UNMAR1 AND SANJAY K. SHARMA2
1
University of Mauritius, Department of Chemical and Environmental
Engineering, Faculty of Engineering, Réduit, Mauritius; 2 Jaipur Engineering
College & Research Centre, JECRC Foundation, Jaipur, Rajasthan, India
11.1 Introduction
A significant majority of synthetic polymers are recalcitrant to any microbial
attack1–3 due to their excessive molecular mass, high number of aromatic rings,
unusual bonds or halogen substitutions.4 Once such a material enters the natural
ecosystem, the harmful effects are long-lasting and give way to several other
hygienic, health and environmental complications. This explains clearly why
plastics then contribute considerably to the contamination of the environment.2
Living conditions in the biosphere have consequently changed drastically and to
such an extent that the presence of non-biodegradable residues is affecting the
survival of many species.5 With the development of technologies for physical,
energetic and chemical recycling of polymeric waste progressing in parallel, an
increasing interest through intensive research and development is now being
devoted to the synthesis and characterization of polymers with enhanced
sensitivity and susceptibility to biodegradation. Thence, the use of biodegradable
332
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334 Chapter 11
14
variety of microorganisms. They tend to be used mainly in the countries where
they have been developed, although there is considerable overlap between the
standard procedures of different countries. This commonness hence provides
wide scope for the development of consistent and international standards.
The growth of composting, a proven sustainable bioremediation technique, as
an ecologically sound waste management technique6 supports the need
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336 Chapter 11
article in the ‘The Chemical Engineer’ magazine (March 2009, Issue 813, p. 6,
Biodegradable PET bottles are ‘‘green breakthrough’’, United Kingdom) high-
lights the development of a form of polyethylene PET which is biodegradable
in both aerobic and anaerobic conditions within five years (ENSO BOTTLES,
a company based in Phoenix, Arizona). The researchers who worked on this
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00332
PET project have modified the PET polymer chain with organic compounds
and microbial nutrients thereby making the polymer structure weak and more
susceptible to faster microbial breakdown.
11.2.2.2 BS series
BS 8472 Method for the determination of compostability (including
biodegradability and eco-toxicity) of packaging materials based on oxo-
biodegradable plastics.
BS EN ISO 14855-2:2009 Determination of the ultimate aerobic bio-
degradability of plastic materials under controlled composting conditions.
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338 Chapter 11
25,29
or mineralization of the polymer). The carbon balance approach, weight
loss analysis, microscopy, spectroscopy and thermal analyses are techniques
that, when combined, deepen the understanding of the degradation of biode-
gradable polymers.25 The degradability of biodegradable polymers is pre-
determined by their chemical and physical structure.2 Of specific importance is
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340
Mohee and Willow Ridge Plastics – PDQ-H Controlled and natural composting Cumulative CO2 evolution for Plastic A
Unmar46 additive (Plastic A) and Ecosafe environments. was much higher than that for Plastic
Plastic – Totally Degradable B. Plastic A therefore showed a higher
Plastic Additives (TDPA) level of biodegradation in terms of CO2
additive (Plastic B). evolution than Plastic B.
Another experiment was undertaken to
observe any physical change of Plastics
A and B as compared to a reference
plastic, namely, compostable plastic
bag (Mater-Bi product – Plastic C).
Thermophilic temperatures were
obtained for about 3–5 days of
composting.
After 55 days of composting, Plastic C
degraded completely while Plastic A
and Plastic B did not undergo any
significant degradation. It was con-
cluded that naturally based plastic
made of starch would degrade com-
pletely in a 60 days.
Mohee et al.47 Mater-Bi Novamont (MB) and Biodegradability was assessed under A biodegradation of 27.1% on a dry
Environmental Product Inc. aerobic and anaerobic conditions. basis for MB plastic within a period of
(EPI). Cellulose filter papers For aerobic conditions, organic 72 days of composting.EPI plastic did
(CFP) were used as a positive fractions of municipal solid wastes not biodegrade under either condition.
control. were composted. For the anaerobic
process, anaerobic inoculum from a
wastewater treatment plant was
used.
Chapter 11
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Kim et al.48 Poly(butylene succinate) (PBS) Natural and aerobic compost soil Weight loss and the reduction in
and bioflour, PBS biocomposite environments. mechanical properties of PBS and the
filled with rice-husk flour (RHF) biocomposites in the compost soil
reinforcing. burial test were significantly greater
than those in the natural soil burial test.
The biodegradability of the biocompo-
sites was enhanced with increasing bio-
flour content because the bioflour is
easily attacked by microorganisms.
Vikman et al.49 New starch-based biopolymers: Enzymatic hydrolysis was performed The enzymatic method is a rapid means
commercial starch-based mate- using excess Bacillus licheniformis of obtaining preliminary information
rials and thermoplastic starch a-amylase and Aspergillus niger about the biodegradability of starch-
films prepared by extrusion from glucoamylase at 37 1C and 80 1C. based materials.
glycerol and native potato
starch, native barley starch, or
cross-linked amylomaize starch.
Gu et al.50 Cellulose acetate (CA) films with In-laboratory composting testing The CA 1.7- and 2.5- DS films com-
degree of substitution (DS) vessels conditions at 53 1C. pletely disappeared by the end of 7-
values of 1.7 and 2.5. and 18-day exposure time periods in
Degradation of Biodegradable and Green Polymers
342
mixing methylated-cornstarch MCS/PVA film were investigated. that the biodegradability of the MCS/
(MCS) with PVA. PVA film strongly depended on the
starch proportion in the film matrix.
The degradation rate of starch in the
starch/PVA film was hindered by
blending starch with PVA.
343
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344 Chapter 11
metabolic activities taking place during the biodegradation, as depicted by
Equation 11.1.57
C6 H12 O6 ðaqÞ þ 6O2 ðgÞ ! 6CO2 ðaq=gÞ þ 6H2 OðlÞ DG ¼ 2829:86 kJ mol1
ð11:1Þ
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00332
This thermophilic stage is governed by the basic principles of heat and mass
transfer and by the biological constraints of living microorganisms.64 Published
data on the rate of heat production by organic material decomposition suggest
that there are wide variations in the rate of heat released by composts. This is
because the rate of heat production is a function of the chemical, physical and
biological properties of the composting materials and substrates.63,65,66
Decomposition by microorganisms during composting occurs predominantly
in the thin liquid biofilms on the surface of the organic particles or other
suitable substrates.57 If the moisture content drops below a critical level, which
is normally 25–30%, the microbial activity will decrease and the micro-
organisms will become dormant. On the other hand, if the moisture content
goes above 65%, oxygen depletion and losses of nutrients through leaching
may occur,65 again lowering the biodegradation rates.
11.3.4 Vermicomposting
One variant of the composting process is vermicomposting whereby earth-
worms are used in the degradation of the substrates. Due to their biological,
chemical and physical actions, earthworms can be directly employed within
bioremediation strategies to promote biodegradation of organic substrates.76
In the vermicomposting process, a major fraction of the nutrients contained in
the organic matter is converted to more bioavailable forms. The first step in
vermicomposting occurs when earthworms break the substrate down to small
fragments as a prelude to ingesting the substrate.75,77 This increases the surface
area of the substrate, facilitating microbial action. The substrate is then
ingested and goes through a process of ‘digestion’ brought about by numerous
species of bacteria and enzymes present in the worms’ gut.75 During this
process, important nutrients such as nitrogen present in the feed material are
converted into forms that are much more water-soluble and bioavailable than
those in the parent substrate.78,79 The earthworms derive their nourishment
from the microorganisms that grow upon the substrate particles. At the same
time, they promote further microbial activity in the residuals so that the faecal
material that they excrete is further fragmented and made more microbially
active than what was ingested as the parent substrate.75
346 Chapter 11
and unsafe to allow for an uncontrolled and unmonitored biodegradation.6 In
this respect, new bioremediation processes that can ‘catalyse’ the degradation
of these wastes in a ‘green’ and controlled manner are needed.6,80 By virtue of
its biochemical reactions that bring about degradation of organic matter,
composting is one such process which is ecologically sound for the degradation
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11.4.1 Polyhydroxyalkanoates
Polyhydroxyalkanoates (PHAs) are bacterial storage polyesters currently
receiving much attention due to their synthesis from renewable resources and
their applicability as biodegradable and biocompatible plastics.87–89 PHAs are
energy/carbon storage materials accumulated under unfavourable growth
conditions in the presence of excess carbon source.90 PHAs are attracting much
attention as substitutes for non-degradable petrochemically derived plastics
because of their similar material properties to conventional plastics and their
complete biodegradability in the natural environment upon disposal.90 Most
aliphatic polyesters are readily mineralized by a number of aerobic and
anaerobic microorganisms that are widely distributed in nature. However,
aromatic polyesters are more resistant to microbial attack than aliphatic
polyesters.91 The fungal biomass in most soils generally exceeds the bacterial
biomass92 and thus it is likely that the fungi may play a considerable role in
degrading polyesters, just as they predominantly perform the decomposition of
organic matter in the soil ecosystem.89
PHAs can be rapidly and completely degraded in municipal anaerobic sludge
by various microorganisms. PHAs have always been found to be biodegrad-
able, but the degradation rate varies with their specific properties.93–95 PHAs
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348 Chapter 11
compost by measuring changes in physical and chemical properties over a
period of 125 days.110 It was found that the incorporation of PEO into starch-
PHBV blends had a negligible effect on the rate of weight loss, while starch in
blends degraded faster than PHBV and it accelerated PHBV degradation. PHB
and PHV moieties within the copolymer degraded at similar rates, regardless
of the presence of starch, as determined by 1H NMR spectroscopy.110 SEM
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O O
C
CH3
O catalyst H H
O C C O C C
heat
O
CH3 CH3 CH3 n
C
poly(lactide)
O
Lactide
to a variety of other common polymers. Therefore, the first issue that needs to be
addressed is the environmental impacts associated with PLAs as regards its
biodegradability.
The disposal of PLA polymeric packaging residues in composting facilities
can be an important method of reducing the amount of packaging materials
that are disposed as municipal solid waste. PLAs are also being engineered to
degrade in composting environments. Real composting conditions differ from
the simulated ones because of factors such as weather, microbial growth and
pH.116 Kale et al.116 assessed the compostability of two commercially available
PLA packages (a bottle and a tray) in real composting conditions. The
degradation of a PLA bottle composed of 96% L-lactide and 4% D-lactide
with bluetone additive and a tray composed of 94% L-lactide and 6% D-lactide
were evaluated in a composting pile having temperatures around 65 1C, a
relative humidity of 65% wet weight moisture content and a pH of 8.5. The
packages were placed in compost in duplicate sets and were taken out on 1, 2, 4,
6, 9, 15, and 30 days. The molecular weight and the glass transition, melting
and decomposition temperatures were monitored to assess the changes in the
packages’ physical properties. After 4 days of being in the compost pile, an
initial fragmentation of the packages was observed. At 15 days of the com-
posting process, the trays started to become a part of the compost whereas the
bottles showed slower degradation and started breaking apart. After 4 days for
the trays and at 6 days for the bottles, a molecular weight reduction of 77% and
85% were observed, respectively. The difference between the degradation times
between the PLA bottles and trays was attributed to their initial difference in
crystallinity. Kale et al.116 reported that the PLA bottles and trays had however
degraded much faster under the ‘real’ composting conditions than in previous
studies of simulated composting conditions.
The composting of extruded PLA in combination with pre-composted yard
wastes in a laboratory composting system was studied by Ghorpade et al.118
Yard waste and PLA mixtures containing 0%, 10% or 30% PLA (dry weight
basis) were placed in composting vessels for four weeks. The data sets for the
monitoring indicated that microbial degradation of PLA had occurred and
there was no significant difference (P 4 0.05) in CO2 emission between the 0%
and 30% PLA mixtures. Based on the observed pH variations, Ghorpade
et al.118 deduced that for 30% PLA, substantial chemical hydrolysis and lactic
acid generation had occurred. The then lowered pH likely suppressed any
further microbial activity. Supported by the GPC results showing a significant
decrease in PLA molecular weight as a result of composting, Ghorpade et al.118
concluded that PLA can be efficiently composted when added in small amounts
of less than 30% by weight to pre-composted yard waste. Gattin et al.119 also
studied the biodegradation of a co-extruded starch/PLA polymeric film in
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350 Chapter 11
liquid, inert solid and composting media. It was found that, irrespective of the
biodegradation medium used, the percentage of mineralization was better than
the required 60% value for the definition of a biodegradable material. The
presence of starch was found to have facilitated the biodegradation of the PLA
component, especially in liquid media.
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11.4.3 Polyethylenes
Polyethylene (PE) is a thermoplastic commodity made by the chemical industry
through the addition polymerization of ethene and which heavily used in a
variety of consumer products. PE is classified into several different categories,
based mostly on its density and branching. The mechanical properties of PE
depend significantly on variables such as the extent and type of branching, the
crystal structure and the molecular weight. The following are types of PE:
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352 Chapter 11
Table 11.2 (Continued )
Reference Substrates Experimental conditions Main results
conditions. Degrada-
tion behaviour of
PLA in natural com-
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sacks, shrink film, packaging film, carrier bags and screw closures.
Low density PE (LDPE) and linear low density PE (LLDPE) are used
predominantly in film applications due to their toughness, flexibility, and
relative transparency.
Very low density PE (VLDPE) is used for hose and tubing, ice and frozen
food bags, food packaging and stretch wrap.
354 Chapter 11
various putrescible wastes and assembled for controlled biostabilization
management under static conditions were used.136 The physical and chemical
deterioration of the polyethylene-starch films exposed to the controlled com-
posting environment were recorded and analysed with respect to the different
composting evolution and were compared with the data collected in pure
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11.4.4 Poly-e-caprolactones
Poly-e-caprolactone (PCL) is a semicrystalline polyester and is of great interest as
it can be obtained by the ring opening polymerization (ROP) of a relatively cheap
monomeric unit ‘e-caprolactone’.25 Scheme 11.2 shows the synthesis of PCL.
PCL is also highly processible as it is soluble in a wide range of organic solvents,
has a low melting point (55–60 1C) and a glass transition temperature of –60 1C
while having the ability to form miscible blends with wide range of polymers.25
PCL undergoes hydrolytic degradation due to the presence of hydrolytically
labile aliphatic ester linkages142 but the rate of degradation is rather slow and may
vary from 2 to 3 years depending on several factors mentioned earlier in this
chapters. PCL is susceptible to microbial degradation143 and is degradable in
several biotic environments,144 including river and lake waters, sewage sludge,
farm soil, paddy soil, creek sediment, roadside sediment, pond sediment and
compost.145 Table 11.3 presents some studies where the biodegradability of PCL
has been studied. It has been reported that esterase and other kinds of lipase could
degrade PCL.146 The degradation times of PCL have also been shown to vary
with molecular weight, crystallinity degree and morphology.20,143,144
Biodegradation of PCL was examined by measuring the release of CO2 when
the polymer was mixed with dog food (used as a model fresh waste) under
controlled laboratory composting conditions.154 It was found out that the
quantity of PCL decomposition, evaluated as the difference in the quantity of
CO2 evolution in the presence and absence of PCL, was in proportion to the
PCL mixing level. The percentage of PCL decomposition, which is calculated as
a ratio of the quantity of PCL decomposition to the mixing level of PCL, was
84% after 11 days in the composting using dog food, but was 59% after the
O O
catalyst
O (CH2)5 C
O
heat
n
e-caprolactone poly(e-caprolactone)
356 Chapter 11
Table 11.3 Research studies on the biodegradability of PCL
Reference Substrates Biodegradation conditions
conditions and the rate of fungal colonization when samples were used as a sole
carbon source for fungus (A. niger). It was found that the inherent biodegrad-
ability of host polymer was enhanced with surface compatibilization156 during
composting for longer incubation. It was observed that the weight loss during
composting increased with the decrease in interfacial tension between filler and
polymer. In general, Singh et al.156 concluded that the inherent biodegradability
of the PCL blends does not depend very significantly on the concentration of
starch in the polyester matrix, but on the compatibilization efficiency.
The biodegradation of polypropylene (PP) (chosen as a non-degradable
plastic), poly(L-lactic acid) (PLLA) and poly(butylene succinate) (PBS) (chosen
as slowly degrading plastics) and polycaprolactone (PCL) and poly(butylene
succinate-co-adipate) (PBSA) (chosen as easily degradable plastics) were tested
in compost made with animal fodder.157 It was observed that the biodegradation
of PLLA and PBS depended on their shape all through the biodegradation tests
while the shapes of PCL and PBSA exerted some deal of influence on their
biodegradability only at the early stage of the biodegradation reactions, while at
the late stage, the biodegradation proceeded almost independently of their shape
in the compost matrix. PCL, poly(vinyl alcohol) (PVAI) and their blends had
been incubated in the presence of a pure strain of microorganisms isolated from
an industrial compost for household refuse.158 In the conditions used, pure PCL
films were completely assimilated over periods of 600–800 hours, whilst
pure PVAI could not be degraded even over much longer exposure times.
Unexpectedly, the blends, even PCL rich, were not altered in the presence of the
microorganisms. De Kesel et al.158 showed that the inactivation of the strain by
PVAI did not occur and was hence not responsible for the lack of degradability.
PVAI, even when present in small amounts in the incubation medium, was
adsorbed on the surface of PCL or blend films thereby rendering the PCL is
inaccessible to the microorganisms for degradation. Šašek et al.2 have tested the
biodegradation of poly(ester-amide)s (prepared by the anionic copolymerization
of e-capro-lactam and e-caprolactone) and aromatic-aliphatic copolyesters
based on glycolysed polyethylene terephthalate from used beverage bottles and
e-caprolactone during composting under controlled conditions and treatment
with ligninolytic fungi. Both methods resulted in the degradation of the
copolymers, composting being more robust.
358 Chapter 11
facilities. In the same area of green waste management, this diversification of
feedstocks could be extended to include all sorts of compostable, and hence
biodegradable, polymeric materials and items comprising packaging films,
bottles and jars, foam packaging, food-service items and consumer products.
This review has summarized and pondered on a variety of research studies with
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00332
Acknowledgements
The authors wish to express their gratitude to all the researchers whose valuable
research findings and discussions have been used in writing this chapter. Our
apologies are nevertheless due to others whose work could not be reported due
to lack of space. Finally, the authors are thankful to other colleagues and the
anonymous reviewers whose suggestions and criticisms have helped improve
this chapter.
References
1. F. Kawai, Adv. Biochem. Eng. Biotechnol., 1995, 52, 151.
2. V. Šašek, J. Vitásek, D. Chromcová, I. Prokopová, J. Brozek and
J. Náhlı́k, Folia Microbiol., 2006, 51, 425.
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34. L. Averous, J. Macromol. Sci. C Polym. Rev., 2004, 44, 231.
35. V. A. Alvarez, R. A. Ruseckaite and A. Vázquez, Polym. Degrad. Stab.,
2006, 91, 3156.
36. A. Keller, D. Bruggmann, A. Neff, B. Müller and E. Wintermantel,
J. Polym. Environ., 2000, 8, 91.
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362 Chapter 11
Iordansakii and G. A. Bonartseva, Commun. Curr. Res. Educs. Topics
Trends Appl. Microbiol., 2007, 295.
88. A. Steinbüchel, Polyhydroxy Alkanoic Acids, ed. D. Byrom, Stockton,
New York, 1991, p. 124.
89. S. Reddy, M. Thirumala and S. Mahmood, J. Microbiol., 2008, 4.
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364 Chapter 11
141. G. J. L. Griffin, J. Polym. Sci.: Polym. Symp., 57, 281.
142. J. C. Middleton and A. J. Tipton, Biomaterials, 2000, 21, 2335.
143. P. Jarrett, C. V. Benedict, J. P. Bell, J. A. Cameron and S. J. Huang,
Polymers as Biomaterials, ed. S. W. Shalaby, A. S. Hoffman, B. D. Ratner
T. A. Horbett, Plenum Press, New York, 1991, p. 181.
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Biodegradable Polymers:
Research and Applications
X. W. WEI, G. GUO, C. Y. GONG, M. L. GOU AND
ZHI YONG QIAN
Sichuan University, West China Medical School, West China Hospital, State
Key Laboratory of Biotherapy and Cancer Center, 610041, Chengdu, China
12.1 Introduction
As we enter the twenty-first century, research at the interface of polymer
chemistry and materials science has given rise to a favourable shift from bio-
stable materials to biodegradable polymers in various fields. Biodegradable
polymers that are either hydrolytically or enzymatically degradable play an
enormous role in industrial, biomedical and other related areas.1 Biodegrada-
tion is a natural process by which organic chemicals in the environment are
converted to simpler compounds, mineralized and redistributed through
elemental cycles such as the carbon, nitrogen and sulfur cycles.2 Biodegradation
is a term that everyone understands, yet no definition has been coined that is
universally acceptable for biodegradable polymers. One of the major reasons
for this is the wide range of disciplines, represented by, namely, biologists,
biochemists, polymer chemists, legislators, environmentalists and manu-
facturers etc. The ones who are involved directly in biodegradable polymer
research or with an opinion on requirements have different opinions on what
they expect a polymer to do in the environment in order to be called or defined
as biodegradable. Mainly, biodegradation of polymeric biomaterials involves
cleavage of hydrolytically or enzymatically sensitive bonds in the polymer
365
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366 Chapter 12
3
leading to polymer erosion. Here we define biodegradable polymers as
polymers that are degraded and catabolized eventually to carbon dioxide and
water under hydrolytic or enzymatic reaction.
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(1) Surgical fixation, such as sutures, clips, bone pins and plates.
(2) Controlled drug delivery (both localized and targeting systems), such as
microspheres and nanoparticles.
(3) Plain membranes for guided tissue regeneration.
(4) Multifilament meshes or porous structures for tissue engineering.
(5) Specialty packaging, such as packaging materials for pharmaceutical
products, drugs and wound dressings.12,13
368 Chapter 12
The work conducted currently in developing various biodegradable materials
is basically some investigation on polymer synthesis. To summarize, polymers
with required properties for specific use are developed as follows.
OH
O
Starch HO
OH
O
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OH
O
Cellulose O
HO
OH
substitution reactions and the C–O–C bond for chain breakage. The nucleo-
philic hydroxyl group of glucose can achieve various properties by reacting
with appropriate polymers or monomers.17 The acetylation of starch is an easy
and well-known example, the obtained starch acetate has several advantages
over native starch, such as improved solubility and better retention of tensile
properties in aqueous environments.18,19
Another field of starch converted to thermoplastic material has caught much
attention. The so-called starch plastics offer an interesting alternative for syn-
thetic polymers achieving rapid degradation instead of long-term durability.20
Thermoplastic starch is generally processable as a traditional plastic; however,
its sensitivity to humidity restricts it to some applications, including loose-fil-
lers, expanded trays, shape moulded parts — mainly as a replacement for
polystyrene.21,22
Starch and its derivatives are also favourable in pharmaceutics being an
alternative for microcrystalline cellulose as an excipient intended for the pro-
duction of pellets via extrusion-spheronization.23 Dukić et al.24 reported the
application of a specific grade of modified starch for extrusion-spheronization
purposes: a crystalline, high-amylose starch formed by gelatinization of amy-
lose-rich starches, followed by enzymatic debranching of amylopectin mole-
cules and retrogradation of linear amylose chains, produces a potential
candidate.24
370 Chapter 12
repeating units. These units are linked by acetal bonds formed between the
hemiacetal carbon atom, C1, of the cyclic glucose structure in one unit and a
hydroxyl group at the C3 atom in the adjacent unit. There exist two isomers due
to the cyclic form, the a-isomer with an axial –OH group on the ring or the
b-isomer with an equatorial –OH group. The only structural difference between
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amylose and cellulose is that starch forms the b-form while cellulose exists in
the b-form (Scheme 12.1). And as a result, enzymes related to acetal hydrolysis
reactions in the biodegradation of each of these two polysaccharides are
different. Cellulose is mainly composed of very long repeating units, thus
making it a highly crystalline, high molecular weight polymer. It is hardly
fusible and soluble in any solvents, except the most aggressive, hydrogen
bond-breaking ones. Due to these intrinsic characteristics, cellulose is usually
converted into derivatives to make it more processable.
Cellulose acetate (CA) is one of the potentially useful cellulose esters, which
is now commercially available. CA of various degree of substitution (DS) are
now being widely used as films and coatings. CA films have a tensile strength
comparable to polystyrene, which makes the polymer suitable for injection
moulding.
Recently, cellulose nanofibres were prepared by fermentation, yielding a very
pure cellulose product with unique physical properties, which distinguished it
from plant-derived cellulose. Its fibres had a high aspect ratio and had a high
surface area per unit mass. This property, when combined with its very
hydrophilic nature, resulted in very high liquid loading capacity. It was an
attractive candidate for a wide range of biomedical and biotechnology
applications.25
OH
O
Chitin
HO O
NHAc
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OH
O
Chitosan
HO O
NH2
Chitosan and its derivatives are suitable for tissue engineering applications
due to their porous structure, gel-forming properties and ease of chemical
modification. There is an excellent review on chitosan in responsive and in situ-
forming scaffolds for bone tissue engineering applications by Martins et al.28
And another new class of biocompatible and biodegradable composite
hydrogels derived from chitosan and oxidized hyaluronic acid was reported.29
The hydrogel was formed upon mixing without the addition of a chemical
cross-linking agent and was able to support cell survival.
Chitosan is one of the most reported non-viral naturally derived polymers for
gene delivery. As a polycation, it has strong affinity for DNA and can spon-
taneously form microspheric particles via complex coacervation.30 One study
on skin delivery of antisense oligonucleotides (AsODNs), which has exciting
potential in the treatment of skin diseases, was recently achieved by AsODN-
loaded chitosan nanoparticles. AsODN-loaded chitosan nanoparticles were
topically applied to Sprague Dawley rats. Animal skin samples were taken for
measurement of beta-galactosidase (beta-Gal) expression and histological
control. After topical application of AsODN-loaded chitosan nanoparticles in
different doses, beta-Gal expression reduced significantly. The results indicates
that chitosan nanoparticles are a useful carrier for delivery of AsODNs into
skin cells of rats and may be used for topical application on human skin.31
372 Chapter 12
functionality of gelling makes alginic acid a favourable biodegradable material
in controlled release drug delivery systems.11
A study on dual-functional alginic acid hybrid nanospheres for cell imaging
and drug delivery has been carried out.32 Gold nanoparticle-encapsulated
alginic acid poly(2-(diethylamino)ethyl methacrylate) monodisperse hybrid
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00365
12.2.5 Collagen
Collagen is considered to be a favourable natural polymer for biomedical use
since it is a major natural constituent of connective tissue and a major struc-
tural protein of any organ. The current main sources of collagen are animal
skin, mostly bovine or porcine, and the properties of collagen, such as
mechanical strength, fluid absorption volume or haemostatic activity, differ
depending on the species and locations of the organisms. Collagen is unique in
its different levels of structural order: primary, secondary, tertiary and
quaternary. And two types of covalent cross-linking, intramolecular and
intermolecular, means that collagen molecules can form fibres in vivo. Bioma-
terials made of collagen are welcomed for several advantages: they are bio-
compatible and non-toxic and have well-documented structural, physical,
chemical, biological and immunological properties.
Due to its biocompatibility and well-established safety profile, collagen
represents a favourable matrix for on-site drug delivery. Ruszczak et al.35
summarized some of the developments and applications of collagen as a
biomaterial in drug delivery systems for antibiotics, where the efforts in the use
of collagen and collagen-synthetic polymer composites for controlled drug
delivery as well as collagen-based diffusion membranes for prolonged drug
release have also been included.35 They assumed that the next generation of
collagen drug delivery system for antibiotics would be focused on both drug
combination and different release profiles which would lead to better infection
control.
Also, research on collagen in the field of tissue engineering was reported.
Oliveira et al. created a transient cartilage template in vitro, which could serve
as an intermediate for bone formation by the endochondral mechanism once
View Online
12.2.6 Gelatin
Gelatin, composed of 19 amino acids joined by peptide linkages, can be
hydrolysed by numerous proteolytic enzymes to yield its constituent amino
acids or peptide components.38
The present cancer gene therapy using gelatin is lacking in both efficiency and
specificity in comparison with viral vectors. However, recent studies showed
that complexes of therapeutic DNA with modified gelatin would possibly offer
a safe and efficient strategy for systemic administration of therapeutic genes to
solid tumours compared to injection of naked plasmid DNA. The in vivo and in
vitro studies using gelatin for the delivery of therapeutic genes to cancerous cells
have been summarized.39
Despite the application in drug delivery systems, the contribution of gelatin
capsules to the control of soil-borne pests was also investigated.40 The gelatin
capsule (gel cap) formulation of 1,3-dichloropropene (1,3-D) has been a new
concept to reduce the environmental release, transport and hazard potential
of the use of 1,3-D to control soil-borne diseases and nematodes. The biological
efficacy of the 1,3-D gel cap formulation under laboratory and greenhouse
trial conditions has been evaluated. Results showed that 1,3-D gel cap
application was as effective as 1,3-D liquid injection treatment on tomato
and crops.
374 Chapter 12
Dextran, a biocompatible, water-soluble polysaccharide, was modified for
specific uses in biomedical research. The modification was performed on by
making its hydroxyl groups into acetal moieties, such that it became insoluble
in water but freely soluble in common organic solvents, thus enabling its use in
the facile preparation of acid-sensitive microparticles.41 Both hydrophobic and
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00365
hydrophilic cargoes were successfully loaded into these particles. The protein
ovalbumin (OVA) has been used in a model vaccine application. Also, work on
hydrogel based on interpenetrating polymer networks of dextran and gelatin
for vascular tissue engineering has been carried out.50
Fibrin has a particular use in suture-less surgery. Recent developments have
established fibrin glue for tissue adhesives as attractive alternatives to sutures.
The current available information on fibrin glue is discussed by Panda et al.46
of different nature and size can be blended with PLA, thus generating a new
class of nanostructured biomaterials or nanocomposites with interesting phy-
sical properties and applications. In addition biocompatible materials such as
carbon nanotubes, cellulose nanowhiskers, hydroxyapitite, etc. could also be
incorporated into the PLA matrix, which increase the potential of PLA for
biomedical applications.
12.3.1.2 Poly(lactide)
The most commercially viable material to date is poly(lactide) (PLA). PLA is
mostly synthesized by the ring-opening polymerization of lactide, which itself
derives from renewable resources such as potato, corn and sugar beet (Scheme
12.3).56 Lactide is a chiral molecule and exists in two optically active forms:
L-lactide and D-lactide. The polymerization of both monomers leads to the
formation of semi-crystalline polymers. However, the polymerization of
racemic (D,L)-lactide and mesolactide results in the formation of amorphous
polymers.57 The particular advantage of PLA polymers is its hydrolysis to lactic
acid, which is one of the metabolites in the carboxylic acid cycle.58 For its
biodegradability and biocompatibility, it was approved by FDA as far back as
the 1970s. PLA was favourable as a material in different fields for many
advantageous physical properties, such as good mechanical properties,
transparency, thermal stability, oil resistance and gas impermeability, as well as
easy processing.59
Copolymers of PLA and poly(ethylene glycol) (PEG) are attractive for
application in drug delivery systems. Novel thymopentin (TP5) release systems
prepared from bioresorbable PLA-PEG-PLA hydrogels were reported
O O
O
* n*
O O O
Glycolide Poly(glycolide)
CH3 O O CH3
O
* n*
O O CH3
O
Lactide Poly(lactide)
376 Chapter 12
60
recently. Bioresorbable hydrogels were obtained by mixing PLLA-PEG-
PLLA and PDLA-PEG-PDLA aqueous solutions due to stereocomplexation
between PLLA and PDLA chains. Thymopentin was taken as a model drug to
evaluate the potential of PLA-PEG-PLA hydrogels as carrier of hydrophilic
drugs. The release profiles were characterized by an initial burst followed by
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00365
slower release. Higher copolymer concentration led to a slower release rate and
less burst effect due to more compact structure which disfavoured drug diffu-
sion. Furthermore, in vivo studies proved the potential of TP5-containing
hydrogels, especially those with a concentration of 25%, which combined with
related results indicated the immunization efficacy of the TP5 release systems
based on PLA/PEG hydrogels. In another study related to nanoparticles of
PLA/PEG copolymer, three types of nanoparticles were formulated using
different block copolymers AB, ABA and BAB (where ‘A’ is PLA and ‘B’ is
PEG) encapsulating hepatitis B surface antigen (HBsAg) to evaluate their
efficacy as an oral vaccine delivery system. Results indicated the efficacy of BAB
nanoparticles as a promising carrier for oral immunization.61
12.3.1.3 Poly(lactide-co-glycolide)
Poly(lactide-co-glycolide) (PLGA) was obtained from copolymerization of
lactide and glycolide, and its structure is shown in Scheme 12.4. The product
exhibits better properties than PLA and PGA. Compared to PGA, PLGA
usually exhibits lower crystallinities and Tm values.
Bulk erosion of PLGA occurred under hydrolysis of the ester bonds. The
degradation rate of PLGA was more rapid than that of PLA and could be
increased by increasing GA content.62 The rate of degradation depended on a
variety of parameters including the LA/GA ratio, molecular weight, and the
shape and structure of the matrix. The popularity of the copolymer accounted
to its good processability and controllable degradation rates. PLGA demon-
strated good cell adhesion and biocompatibility, which made it a potential
candidate in both tissue engineering and micron-/nano- drug delivery
systems.63
Recently, investigation on poly(lactide-co-glycolide) implants was carried
out, aiming at assessing the feasibility of hot-melt extrusion (HME) for pre-
paring implants based on PLGA formulations with special emphasis on protein
stability, burst release and release completeness. The results demonstrated that
the release from all implants reached the 100% value in 60–80 days with nearly
O H
HO x O y
12.3.1.4 Polycaprolactone
Polycaprolactone (PCL) is a semi-crystalline, hydrophobic polymer with a
relatively polar ester group and five non-polar methylene groups in its repeating
unit. It is mostly synthesized by the ring-opening polymerization method from
e-caprolactone (e-CL) monomers. The high olefin content imparts polyolefin-
like properties to PCL. However, owing to the high degree of crystallinity and
hydrophobicity, PCL degrades rather slowly and is less biocompatible with soft
tissue, which restricts its further clinical application.66 Therefore, the mod-
ification of PCL is proposed.67–69 Poly(ethylene glycol) (PEG), chosen for its
hydrophilicity, non-toxicity and absence of antigenicity and immunogenicity,
can be attached to PCL, forming PCL/PEG copolymers (Scheme 12.5).69–77
The PCL/PEG copolymers can be summarized into several types including
PCL-PEG diblock copolymer, PCL-PEG-PCL (PCEC), or PEG-PCL-PEG
(PECE) triblock copolymers and star-shaped polymers. For the synthesis and
applications of each type one can refer to a recent review by Wei et al.78 The
PCL/PEG copolymers have various applications in biomedical uses, particu-
larly, the triblock copolymers of PCEC and PECE found potential as in situ
gel-forming hydrogel drug delivery system.
Gong et al. have made a series of investigations on PCL/PEG triblock
copolymers.69,75–77 Aqueous solution of PCEC copolymer displayed thermo-
sensitive sol–gel–sol transition behaviour, which is a flowing sol at low tem-
perature and turns into a non-flowing gel at body temperature. The cytotoxicity
of PCEC copolymer was evaluated. In vivo gel-formation, degradation test,
acute toxicity tests and histopathological study of PCEC hydrogels were per-
formed in BALB/c mice by subcutaneous administration, and histopathologic
study was done. The obtained PCEC hydrogel was non-toxic after
O H
O O CH2
CH3O xH + O CH3O x 5 O y
378 Chapter 12
75
subcutaneous administration. In their further study, sol–gel–sol transition
and drug delivery behaviour was further investigated. The sol–gel phase tran-
sition mechanism was investigated using 13C-nuclear magnetic resonance
imaging and a laser diffraction particle size analyser. The in vitro release
behaviors of several model drugs were studied. An anaesthesia assay was
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00365
conducted using the tail flick latency (TFL) test to evaluate the in vivo con-
trolled drug delivery. All results indicated that PCEC hydrogel is promising for
use as an injectable local drug delivery system.77
12.3.1.5 Polyhydroxyalkanoates
The natural polyesters, which are synthesized and accumulated intracellularly
during unbalanced growth by a wide variety of microorganisms, are receiving
increased attention.79 Microbial polyhydroxyalkanoates (PHAs) is one of the
largest groups of these polyesters, and is the most appropriate candidate for
biodegradable plastics.80 The members of this family have the general structure
given in Scheme 12.6. All of these polyesters contain units which are 100%
optically pure at the b-position and their material properties varies from rigid
brittle plastics, to flexible ones and to strong elastomers with the variation in
size of pendant alkyl group and composition of the polymer. Poly(D-3-
hydroxybutyrate) (PHB), with R ¼ CH3 is the most ubiquitous and most
intensively studied PHA with high crystallinity and relatively high Tg. Con-
siderable interest arose in the synthesize of PHB by controlled fermentation and
chemical synthesis.81 Also, both routes were suitable for the modification of
PHB. Particularly, different copolymers with better mechanical properties than
PHB were successfully obtained by feeding the bacteria with a variety of carbon
sources in fermentation.82
An interesting investigation was made to indicate that PHB can form a new
biodegradable adsorption material. Poly-3-hydroxybutyrate (PHB) was used as
a new material to prepare a biomimetic adsorbent by a modified double
emulsion solvent evaporation technique. The enrichment capacities of the
adsorbent for toxic liposoluble organic compounds were evaluated by chloro-
benzene (CB) and o-nitrochlorobenzene (o-NCB) with the adsorption iso-
therms, enrichment factor (EF) and enrichment kinetics.83
Ye et al. have looked into PHB scaffolds, and worked on the potential of
PHB/poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) (PHB/
PHBHHx) to produce neocartilage upon seeding with differentiated human
adipose-derived stem cells (hASCs). The study demonstrated that PHB/
PHBHHx was a suitable material for cartilage tissue engineering.84
O *
* n CH2
R= *
R O x
Enzymatically
hydrolyzable linkage
Hard segment of
polyurethane
Biodegradation
380 Chapter 12
vessels was simulated, showing that the SMPU stents can be comfortably
implanted while minimizing the overpressure onto the vessel walls, due to their
thermo-responsive shape memory behaviour. More over, the work by Guo
et al. reported that highly adjustable and precisely controllable drug release
from a biodegradable stent coating was achieved using a unique family of
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00365
12.3.3 Polyamides
Although polyamides contain the same amide linkage that is found in poly-
peptides, aliphatic polyamides are generally resistant to microbial and
enzymatic attacks.2 What is responsible for their low biodegradability are the
strong intermolecular interactions caused by hydrogen bonding. Thus,
attempts were made to enhance the biodegradability of aliphatic polyamides
mainly in two ways: (1) the introduction of ester linkages;93 (2) the incor-
poration of a-amino acid residues. Poly(ester amide)s is a class of polymers
with both advantages of polyesters and polyamides, and which appears to have
ideal material and processing properties and good biodegradability.94 It can
either be synthesized by polycondensation of ester-containing diamines with
dicarboxylic acids or by ring-opening polymerization of depsipeptides.95 The
presence of hydrolytically readily cleavable ester bonds in the backbone makes
poly(ester-amide)s a promising material for their use in biomedical fields.
Studies on the blood and tissue compatibility of poly(ester amide) co-poly-
mers were carried out. A family of biodegradable poly(ester amide) (PEA)
co-polymers based on naturally occurring a-amino acids has been developed
for applications ranging from biomedical device coatings to delivery of ther-
apeutic biologics. To gain insight into this process, representative elastomeric
PEAs designed for a cardiovascular stent coating were compared to non-
degradable and biodegradable polymers in a series of in vitro assays to examine
blood and cellular responses. These in vitro studies of the blood and tissue
compatibility of these biodegradable, a-amino acid-based PEAs suggested that
they might support a more natural healing response by attenuating the pro-
inflammatory reaction to the implant and promoting growth of appropriate
cells for repair of the tissue architecture.96
12.3.4 Polyanhydrides
Polyanhydrides have been intensively investigated as important biomaterials in
medical fields due to their excellent biodegradability and biocompatibility.
Over the decades, numerous studies have been carried out in academia and
industry which were mainly concerned with chemical and physical character-
ization of these polymers, degradation properties, toxicity studies and various
View Online
12.3.5 Polyphosphoesters
Polyphosphoesters (PPEs) refers to a wide range of biodegradable polymers
with repeating phosphoester linkages in the backbone whose general structure
is shown in Scheme 12.8. The initial application of polyphosphoesters in bio-
medical fields dates back to the 1980s.98 Since then, polyphosphoesters have
been attractive for extensive uses and have been investigated thoroughly
looking at the versatility of the PPE’s pendant groups. The introduction of
bioactive molecules and various modifications can be achieved thanks to the
pentavalent phosphorus atom, thus obtaining PPE with modified physical and
chemical properties. When it comes to the syntheses of PPEs, polycondensation
has been one of the most often used methods. The reactions are usually carried
out between diols and phosphonic dihalides or diallyl phosphoesters.99 There
are also other ways of synthesizing reported, such as polyaddition, transes-
terification and ring-opening polymerization by enzymes and by other
catalysts. For the specific reaction, we can refer to a review by Wang et al.100
Recently, novel thermo-responsive block copolymers of poly(ethylene glycol)
and polyphosphoester were synthesized, and the thermo-induced self-assembly,
biocompatibility and hydrolytic degradation behaviour were studied and its
biocompatibility and thermo-responsiveness were ensured.101 Similarly, aqu-
eous dispersions of thermo-sensitive gold nanoparticles protected by diblock
copolymers of poly(ethylene glycol) and polyphosphoester were prepared and
studied.102 These gold nanoparticles protected by thermo-sensitive diblock
O
* P O R O *
n
R'
382 Chapter 12
copolymers with tuneable collapse temperature are expected to be useful for
biomedical applications.
12.3.6 Others
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00365
(A) OR
O
*
* n
CN
(B) R
* N P *
n
R
12.4 Conclusions
Tremendous progress has been attained in seeking and optimizing biodegrad-
able polymers with appropriated properties in modern industrial applications
and biomedical use. Reported studies on biodegradable polymers currently
involved natural polymers such as cellulose and synthetic polymers such as
polyesters. Owing to the presence of hydrolytic bonds (hydrolytically or
enzymatically), these polymers can both be promising candidates as bioma-
terials with good biocompatibility and an attractive approach to environmental
waste management. Further studies concerned with biodegradable materials
might be facilitated by advances in synthetic organic chemistry and novel
bioprocesses. Although many advances have been made, much work still
remains before a wide range of biodegradable materials will be used commonly
throughout the world, which is earnestly making the world of tomorrow
greener.
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CHAPTER 13
Impacts of Biodegradable
Polymers: Towards Biomedical
Applications
Y. OMIDI AND S. DAVARAN
13.1 Introduction
In the third millennium of mankind’s history, it is now deeply believed that
mimicking the natural process of life at the cellular/molecular level is a key for the
advancement of novel medicines to optimize quality of life and control diseases.
We are now moving fast to develop new generation of nano-biomedicines
with maximal efficiency and minimal inadvertent consequences. To pursue
such an aim, smart and specific approaches are necessary to control aberrant
biofunctionalities of cells/tissues. Of these, one important matter is to avoid the
undesired bioimpacts of materials used for medical interventions. In general,
biocompatibility (i.e. cyto- and/or geno-compatibility) of advanced materials
looms as a key factor. In fact, pharmacokinetically, a xenobiotic is subjected to
ADME (adsorption, distribution, metabolism and elimination) processes. Thus,
to avoid or to lessen toxic impacts, natural ‘biodegradation’ is a keystone of
such a process. Biodegradation is the chemical breakdown of materials by the
physiological environment. The word is often used in relation to ecology, waste
management and environmental remediation, termed as ‘bioremediation’.
388
Impacts of Biodegradable Polymers: Towards Biomedical Applications 389
Basically, the evolution of biodegradable polymers appeared to be through
chain modification of existing materials or by modulating the chemical
composition of synthetic polymers using several polymerization techniques.
Biodegradable polymers with controlled degradation characteristics can be
used as an important part of biomedical sciences such as drug delivery, cell and
tissue engineering, tissue repair, stem cell therapy and molecular detection and/
or therapy.1–3 To date, many types of natural and synthetic biodegradable
polymers, for example albumin, chitosan, and hyaluronic acid and derivatives,
have been investigated for medical and pharmaceutical applications. Use
of natural polymers (e.g. cellulose and starches) appears to be still common
in biomedical research; nevertheless, synthetic biodegradable polymers are
increasingly exploited in pharmaceutical and tissue-engineering products.
Basically, synthetic polymers can be prepared with chemical structures tailored
towards optimized physical properties, as result of which they can be
considered as biomedical materials with well-defined purities and compositions
superior to those attainable when using natural polymers. There exist various
established chemical classes of synthetic biodegradable polymers which offer
good enough biocompatibility. Of note, the rate of biodegradation and
mechanical strength of such advanced biodegradable polymers can be tuned.
From biocompatibility view points, the biological degradation of polymers is
deemed to be very important in biomedicine for many reasons. For instance,
the biologically degradation of a polymeric implant within the body clearly
indicates that surgical intervention may not be required for removal at the end
of its functional life, thus excluding need for any further surgical operation.
Further, in tissue engineering and regenerative medicine, implementation
of biodegradable polymers confers great potential to design such an approach
to approximate tissues through providing a polymer scaffold that can withstand
mechanical stresses and provide an appropriate surface as an extracellular
matrix for attachment and growth of cells. And ideally, it should be degraded at
an appropriate rate, allowing the load to be transferred to the new tissue.
Principally, organic material can be degraded aerobically with oxygen, or
anaerobically without oxygen. Of the biodegradable polymers, bioabsorbable
polymers can be applied for wound closure (sutures, staples), osteosynthetic
materials (orthopedic fixation devices, pins, screw, rods, bone plates), cardiovas-
cular surgery (stents, grafts) and intestinal surgery (anastomosis rings).4 Bio-
absorbable polymers also find applications as matrix materials for implanted
drug release devices or drug-containing microspheres or microcapsules, and drug
delivery vehicles, such as micro/nano-particles, micelles, hydrogels and injectable
delivery systems.
In general, the major biomedical applications of biodegradable polymers5,6
are as follows:
bone replacement
drug delivery using synthetic biodegradable polymers
green biodegradable packaging materials
tissue engineering with synthetic biodegradable polymers
390 Chapter 13
green biodegradable sutures
vascular stents
general and reconstructive surgery.
Figure 13.4 Most commonly used methods for preparation of biodegradable nano-
particles (NPs).
13.3.3.4 Nanoprecipitation
The nanoprecipitation technique (the so-called solvent displacement method)
for preparation of NPs was first developed by Fessi and co-workers.48 In this
method, NP formation is instantaneous and the entire procedure is carried out
in only one step. This method requires two solvents that are miscible, and
ideally both the polymer and the drug must dissolve in the solvent (first system),
but not in the non-solvent (the second system). Nanoprecipitation occurs by a
rapid desolvation of the polymer when the polymer solution is added to the
non-solvent. Once the polymer-containing solvent has diffused into the
dispersing medium, the polymer precipitates – resulting in immediate drug
entrapment. The rapid nanoparticle formation is due to interfacial turbulence
that takes place at the interface of the solvent and the non-solvent and results
from complex and cumulated phenomena such as flow, diffusion and surface
tension variations.48,56 Nanoprecipitation often enables the production of small
nanoparticles (100–300 nm) with narrow unimodal distribution and a wide
range of preformed polymers can be used, such as poly(D,L-lactic-co-glycolic
acids), cellulose derivatives or poly e-caprolactones. This method does not
require extended shearing/stirring rates, sonication or very high temperatures,
and is characterized by the absence of oily-aqueous interfaces, all conditions
that might damage a protein structure. Moreover, surfactants are not always
needed and unacceptable toxic organic solvents are generally excluded from
this procedure.48
Figure 13.5 Schematic representation of single (a, b) and double (c, d) emulsions.
402 Chapter 13
to control the size of the particles from 270 nm to 59 mm by changing the
parameters such as the O/W ratio of the S/O/W emulsion and the stirring
speed. The release rate of hGH appeared to be dependent upon the composition
of Dex-g-PLGA. In vivo studies in mice revealed that the plasma concentration
of hGH was maintained for 1 week without a significant initial burst. Figure
13.6 represents scanning electron microscopy (SEM) and confocal laser
scanning microscopy (CLSM) of the particles. Morphologically, particles
obtained by solvent evaporation of the solid-in-oil-in-water (S/O/W) emulsion
appeared to be spherical with a smooth surface (Figure 13.6a,b), implying that
the free polymer that existed in the organic phase may form a polymer matrix in
which the nanoparticles are dispersed. The S/O/W emulsion method is deemed
to be effective for the stable encapsulation of proteins in particles. In fact, the
solidified protein particles in the S/O/W emulsion seem to suppress the contact
between the protein and the water/oil interface – the phenomenon which is
considered as one of the major causes of denaturation of protein-based drugs.
Using CLSM, Kakizawa et al. showed the distribution of the encapsulated
molecules within the particles (Figure 13.6c), which was confirmed by differ-
ential interference micrographs (Figure 13.6d).80
Figure 13.6 Scanning electron micrographs of (a) nanoparticles in the S/O suspension
and (b) microparticles of Dex-g-PLGA. Confocal microscopy image
(c) and differential interference micrograph (d) of Dex-g-PLGA micro-
particles containing a model compound (FD 40). Dex-g-PLGA: poly
(lactide-co-glycolide)-grafted dextran. (Reproduced with Permission
from Prof. N. Ida. Original work of citation for figure is Kakizawa Y.,
Nishio R., Hirano T., Koshi Y., Nukiwa M., Koiwa M., Michizoe J.
and Ida N., J Control. Release, 2010, 142(1), 8.)
Impacts of Biodegradable Polymers: Towards Biomedical Applications 403
Recently, Cui et al. (2010) investigated the effect of pigment epithelial-
derived factor (PEDF) gene loaded in PLGA nanoparticles (PEDF-PLGANPs)
on mouse colon carcinoma cells (CT26s) in vitro and in vivo.81 These researchers
showed lower cytotoxicity for blank PLGANPs (bPLGANPs) in comparison
with cationic polymers such as polyethylenimine (PEI) in CT26s cells, while the
PEDF-PLGANPs directly induced apoptosis in CT26 cells and inhibited
proliferation in human umbilical vein endothelial cell (HUVEC). In vivo
investigation revealed PEDF-PLGANPs inhibition in CT26 tumour growth
by inducing CT26 apoptosis, decreasing MVD and inhibiting angiogenesis.
These results indicate that PLGANP-mediated PEDF gene could provide an
innovative strategy for the therapy of colon carcinoma.
Furthermore, PLGA micro/nanospheres have been utilized for sustained/
controlled release of various genetic drugs (e.g. plasmid DNA and oligonu-
cleotides). These biopolymers have been shown to improve delivery of DNA to
antigen-presenting cells (APC) by efficient trafficking through local lymphoid
tissue and uptake by dendritic cells.82 Basically, the water-in-oil-in-water
(W/O/W) double emulsion/solvent evaporation method has been successfully
exploited for encapsulation/entrapment of plasmid DNA within PLGA
microspheres. Since plasmid DNA is a highly charged macromolecule, DNA
should be first dissolved in a small volume of an aqueous phase, emulsified in an
oil phase, and then re-emulsified in an aqueous phase under high shear stress
conditions to prepare PLGA microspheres. Nevertheless, such formulation
process may structurally damage the encapsulated plasmid DNA, altering its
structural integrity and biological activity. Besides, polymer degradation can
induce an acidic microenvironment inside eroding PLGA microspheres, upon
which the encapsulated DNA may undergo further structural damage during
the release period.
In 2008, Mok and Park encapsulated plasmid DNA within PLGA nano-
spheres by using the polyethylene glycol (PEG) assisted solubilization tech-
nique on plasmid DNA in organic solvents.83 Plasmid DNA was solubilized in
an organic solvent mixture composed of 80%dichloromethane and 20%DMSO
by producing PEG/DNA nano-complexes (with diameter B100 nm). DNA
could be solubilized in the organic solvent mixture to a greater extent by
increasing the weight ratio of PEG/DNA. DNA from the O/W PLGA nano-
spheres remained intact; however, DNA from the W/O/W PLGA nanospheres
appeared to be completely degraded. In vitro release experiments revealed intact
release of plasmid DNA from the O/W PLGA nanospheres with an initial burst
profile followed by the sustained release – perhaps due to the non-uniform
distribution of PEG/DNA nano-complexes within PLGA nanospheres. For the
rapid release of DNA within 24 h, these researchers discussed that hydrophilic
PEG/DNA nano-complexes entrapped within PLGA nanospheres were pre-
ferentially hydrated and swollen upon incubating in aqueous medium and then
quickly released out by osmotic rupture of surrounding PLGA polymer phase.
Based on microscopic analysis, many PLGA nanospheres were found in the
intracellular area. The cells certainly expressed an exogenous GFP gene,
nonetheless the GFP expression level in the macrophage cells was very low to
404 Chapter 13
quantify, compared to those transfected with commercially available poly-
plexes and lipoplexes. In fact, DNA-loaded PLGA nanospheres could not
efficiently escape from the endosome and lysosome compartments after cellular
uptake, in which incorporation of fusogenic peptides in the PLGA nanospheres
seems to be desirable for an enhanced gene expression efficiency.
In addition to protein- and gene-based macromolecules, PLGA-based micro/
nanosystems have been successfully used for delivery of vaccine.84–86 Ingestion
of intruding pathogens in the body results in migration of dendritic cells (DCs)
from the periphery to the lymph nodes, where processed pathogen-derived
antigens are presented to T cells. Such an interaction between DCs and T cells
controls the type and magnitude of the resulting immune response, as a result
of which some researchers in preclinical and clinical studies have exploited DCs
in an attempt to improve vaccine efficacy. In fact, the efficacy of vaccines is
strongly enhanced by antibody-mediated targeting of vaccine components to
the antigen-presenting DCs.78 However, the options to link a single antibody to
multiple vaccine components (e.g. multiple antigens and immune modulators)
are limited. To overcome such limitation, many researchers have focused on
development and/or advancement of nano- and micrometer-sized slow-release
vaccine delivery systems that specifically target human DCs. For example, in an
interesting study, Cruz et al. (2010) coated the PLGA-based nano- and
microparticles (with diameters B100–1000 nm) with a polyethylene glycol-lipid
layer carrying the humanized targeting antibody hD1, which does not interact
with complement or Fc receptors and recognizes the human C-type lectin
receptor DC-SIGN on DCs.87 The encapsulation of antigen resulted in almost
38% degradation for both nanoparticles and microparticles 6 days after par-
ticle ingestion by DCs, nevertheless the NPs effectively targeted human DCs.
Consequently, targeted delivery only improved antigen presentation of
NPs and induced antigen-dependent T cell responses at 10–100 fold lower
concentrations than nontargeted NPs. The PLGA-PEG particles displayed
approximately 20–30 mg antibody per mg PLGA. Analysis by fluorescence-
activated cell sorting (FACS) flow cytometry and CLSM revealed that
antibodies can present on the PLGA surface.
Recently, we have developed a star-shaped PLGA-b-cyclodextrin (PLGA-
b-CD) copolymer by reacting L-lactide, glycolide and b-cyclodextrin in the
presence of stannous octoate as a catalyst.40 The structure of PLGA-b-CD
copolymer was confirmed with 1H-NMR, 13C-NMR, and FT-IR spectra.
Adriamycin (ADR) as an anticancer agent was encapsulated within the PLGA-
b-CD nanoparticles and effects of the experimental parameters (e.g. copolymer
composition, ADR concentration, copolymer concentration and poly(vinyl
alcohol) concentration) on particular size and encapsulation efficiency were
investigated. Figure 13.7 represents the SEM micrographs particles of PLGA-
b-CD. We found that an increase in the internal aqueous phase volume can
lead to a decrease in particle average size, and a decrease in the polymer
concentration can cause an increase in particle average size (i.e. from 135.5 to
325.6 nm). All of the release profiles indicated a close relationship between
each formulation variable and the amount of ADR released.
Impacts of Biodegradable Polymers: Towards Biomedical Applications 405
Figure 13.8 Selected examples of commonly used synthetic polycationic polymers for
gene delivery. PEI: Poly(ethylenimine); PLL: Poly(L-Iysine); PAMAM:
Polyamidoaminedendrimer.
410 Chapter 13
(e.g. DAB8 and DAB16) can inadvertently induce alterations in gene expres-
sion.104,105 Of these dendrimers, we have previously shown dramatic alteration
in gene expression induced by DAB16 dendrimer in A431 and A549 cells.105
Figure 13.9 represents selected examples of DNA microarray data as the scatter
plots of gene expression profiles induced by various cationic polymers such as
polyamidoamine and linear and branched polyethylenimine in A431 cells.
Importantly, some of the genes altered by these polymers are related to cell
defence and response to stress (e.g. ALOX5, TNFRSF7) and apoptosis (e.g.
TNFRSF7). For example, in A549 cells, some of the altered genes were in
association with cell defence, DNA repaire/damage and apoptosis (e.g. CCNH;
ERCC1; PCNAM, CD14). In A549 cells treated with complexed DAB den-
drimer with DNA (DBA16:DNA nanoparticles), we found expression changes
for some important genes (i.e. TGFb1, BCL2a1, IL5, CXCR4 AND PCKa).
Of these, the BCL2 protein family is involved in a wide variety of cellular
activities that also act, in particular, as anti- and pro-apoptotic regulators. The
protein encoded by this gene is able to reduce the release of pro-apoptotic
cytochrome c from mitochondria and block caspase activation which is the main
apoptosis pathway. The up-regulation of TGFb1 and BCL2a conceivably imply
occurrence of apoptosis upon treatments with DAB16:DNApolyplexes;
nonetheless, cells can also undergo a compensatory stage during apoptosis
phenomena. We found that the altered genes induced by PolyFectt and DAB16
dendrimers in A431 cells shows some commonalities and differences in gene
expression pattern, presumably due to their positive charge and structural
Figure 13.9 Selected examples of scatter plots of gene expression changes induced by
cationic polymers in A431 cells. Data represent Log2 transformed gene
expression values. Above 2-fold change in expression of treated to
untreated is indicated by reference lines, showing over-expressed genes
(bold circles) and unchanged genes (unfilled circles). PF: Polyfectt;
bPEI: branched polyethylenimine; lPEI: linear polyethylenimine (our
unpublished data produced by Omidi et al.).
Impacts of Biodegradable Polymers: Towards Biomedical Applications 411
architecture. In A431 cells, cells treated with either DAB8 or DAB16 resulted in
B13% and B7%similar and opposite patterns of gene expression changes,
respectively. It should be remembered that the identity of the genes whose
expression was significantly altered (i.e. the ‘gene signature’ of the delivery
system) was markedly different in the two cell lines, despite the similar expression
of the majority of the genes (80%) that remained unaffected.106
Likewise, Pluronict block copolymers were shown to cause various
functional alterations in cells through interacting with cellular biomolecules
and thus affecting various cellular functions such as mitochondrial respiration,
ATP synthesis, activity of drug efflux transporters, apoptotic signal transduc-
tion and transcriptional activation of gene expression both in vitro and in
vivo.107 This polymer is able to enhance expression of reporter genes under the
control of cytomegalovirus promoter and NF-KB response element in stably
and transiently transfected mouse fibroblasts and myoblasts in vitro. These
block copolymers acted as biological response modifying agents by up-
regulating the transcription of genes through the activation of selected signal-
ling pathways, in particular, NF-KB.108 Furthermore, Plutonic P85 (P85)
promoted transport of the pDNA to the nucleus in cells transiently transfected
with DNA/PEI polyplex.109 P85 has also been used successfully for DNA
vaccines; however, it was reported that P85 simultaneously increased transgene
expression and activated immunity, in which P85 alone and P85:DNA
complexes were shown to increase the systemic expansion of CD11c+ (DC),
and local expansion of CD11c+, CD14+ (macrophages) and CD49b+ (natural
killer) cell populations. DNA/P85 polyplex can also increase maturation of
local DC (CD11c+ CD86+, CD11c+ CD80+, and CD11c+ CD40+).110
Thus, the activation of immunogenes in the antigen-presenting cells by P85:
DNA complexes can highlight new insights for these kinds of polymers.
It has been shown that Pluronict can cause some alterations in hsp68
expression, suggesting that this polymer may affect stress-related pathways or
there is a cross-talk between the stress and other pathways activated by the
copolymer.108 This finding is in accord with what we have observed for some
other cationic polymers or lipids. Pluronict (Pluronict L61 and Fl27; also
called as SP1017) has been reported to deliver plasmid DNA in skeletal and
cardiac muscle, as well as in solid tumours. Unlike other polycations, Pluronict
does not bind and condense the nucleic acids, it does not protect DNA
from degradation or facilitate transport of the DNA into the cell and its effects
involve transcriptional activation of gene expression.109 The effect of Pluronict
was reported to be related to the activation of gene expression by activating
the NF-KB and p53 signalling pathways, in which pro-apoptotic AP-l gene
that is frequently regulated by the NF-KB system, was not responsive. This,
perhaps, indicates that Pluronic-mediated effect on transcription is selective
and not a result of a general non-specific activation of immune defence system
such as NO-mediated burst. Kabanov’s group has reported that Pluronic-
block copolymers interact with biomembranes and induce gene expression
through mechanisms that differ from the delivery of DNA into the cell.
They also questioned whether up-regulation of expression of genes delivered into
412 Chapter 13
cells can also take place by other non-viral polymer-based gene delivery systems.
We have observed that various polymers, in particular polycations, are able
to affect gene expression related to immune response and cell defence.38,111
PLGA copolymers have been reported as a biocompatible polymer with no
or little inadvertent cellular impacts. To assess such a concept, we have
examined the genocompatibility and toxicogenomics of PLGA in A431 cells
using microarray technology (our unpublished data). Briefly, the A431 cells at
40–50%confluency were exposed to routinely recommended concentration of
PLGA for 4 h. After 24 h, 10mg total RNA was extracted and examined for
yield and purity, then using a reverse transcription reaction it was converted to
cDNA, i.e. aminoallyl-labelled cDNA (aa-cDNA). The aa-cDNA from
untreated and treated cells were post-labelled with cyanine (Cy3 and Cy5) dyes
and after purification were hybridized on a slide microarray housing 200 gene
spots (Ocimum Biosolutions, India). After washing, the slide arrays were
scanned (TECAN, Switzerland) and data were analysed using ArrayPro soft-
ware. Table 13.1 represents some of the up- and down-regulated genes altered
Acknowledgements
The authors are grateful to the Research Center for Pharmaceutical Nano-
technology, Tabriz University of Medical Sciences for financial support.
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CHAPTER 14
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00419
Biodegradable Injectable
Systems for Bone Tissue
Engineering
RICHARD T. TRAN, DIPENDRA GYAWALI,
PARVATHI NAIR AND JIAN YANG
14.1 Introduction
Natural bone has an intrinsic ability to heal itself, and this regenerative capa-
city is diminished with age, illness, or injury.1 The current approaches for the
treatment of critical-sized bone defects include the use of natural bone grafts or
metallic prosthetic implants. Natural grafts can be categorized based upon their
tissue source as autografts, allografts, and xenografts. Autografts currently
serve as the gold standard for bone implantation, and are harvested from the
patient’s own body, which reduces the risk of graft rejection. Although,
autografts are widely used in clinical applications, problems such as donor site
morbidity, the invasive nature of surgery, long recovery times, and bone graft
availability of a desired size and shape have restricted the use of autografts in
orthopedic applications.2,3 In the United States (US), there have been over a
million surgical procedures involving large bone defects due to trauma, non-
union healing fractures, or resection requiring the use of bone grafts. As the US
population ages, there has been an increase in demand for bone grafts, and
these surgical procedures have placed a large burden on the healthcare industry
419
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420 Chapter 14
4,5
totaling over 5 billion dollars annually. Thus, a clinical need exists for the
development of alternative methods to regenerate bone to meet the shortage in
bone grafts and address the limitations of the current available treatment
choices.
Tissue engineering is a multidisciplinary field, which applies principles from
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00419
Figure 14.2 Schematic representing the concept behind injectable bone tissue
engineering.
422 Chapter 14
14.2.1 Injectability and In Situ Cross-linking
The basic requirements for the selection of scaffold material for tissue engi-
neering applications are challenging and multi-faceted. For injectable-based
systems, the precursor solution of the polymer should be easily injectable
through the required delivery system in order to facilitate the minimally inva-
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00419
Table 14.1 Mechanical properties for the various sections of native bone.17
Mechanical strength
Young’s
Bone tissue Tensile Compressive modulus
has been reported that under fluid flow stimulation, osteoblasts have been shown
to reorganize their cytoskeleton, up-regulate transmembrane focal adhesion
proteins, and express osteoblast-specific proteins such as osteopontin involved in
extracellular matrix adhesion.17,22,23 Thus, in order to simulate the dynamic
mechanical physiological environment, injectable materials should possess
similar mechanical properties compared to native bone tissue.
14.2.3 Porosity
The porosity of the scaffold architecture is another important design require-
ment, which plays a critical role in the outcome of a tissue engineered graft.
Cells seeded inside the scaffold rely heavily on the void spaces within the
construct for cellular in-growth, vascularization, and the exchange of nutrients
and waste products.11 Thus, the implanted graft should have a highly orga-
nized, porous, and interconnected structure. Porosities of more than 90% and
pore sizes greater than 300 mm are preferred for cellular penetration and vas-
cularization while maintaining scaffold mechanical integrity.24–28 Recent
research has shown that nanoscale architectures of the scaffold can also
influence the cellular behavior of the underlying material.17 In particular to
injectable materials, in situ porous generating techniques have been limited to
gas foaming29 and particulate leaching.30 The foremost challenge for the suc-
cess of injectable systems still lies in the creation of injectable scaffolds with an
interconnected and homogeneous porous network.
14.2.4 Biodegradation
Tissue engineered devices should be fully resorbed by the body to match the
rate of neotissue formation, and cleared from the body through normal phy-
siological functions through a process known as biodegradation. Biodegrada-
tion is a process by which polymers are chemically reversed into their precursor
monomers, which can be accomplished through dissolution, hydrolysis, and
enzymatic activities.31 Degradation mechanisms for polymers used in tissue
engineering are categorized as either bulk degradation or surface erosion. Most
polyesters, polyether-esters, and polyester-amides rely on bulk degradation and
follow first-order profiles, whereas polyanhydrides and polyorthoesters degrade
through surface erosion and yield zero-order profiles.17
Injectable polymers used as bone scaffolds are composed of cross-linked
networks, which degrade through several mechanisms. Degradation of these
networks primarily relies on the nature and location of the degradable groups
and cross-linkable moieties. In the case of poly(propylene fumarate) (PPF) and
poly(ethylene glycol maleate citrates) (PEGMC), the cross-linkable and
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424 Chapter 14
degradable moieties are found alternating along the polymer backbone chain.
As the polymer network degrades, it is broken down into multiple kinetic
chains and the starting monomers. Lactic acid-based injectable materials are
degraded into the original core molecules connected by the degradable units
and kinetic chains formed during the free-radical polymerization of the
photoreactive groups.12 Finally, enzymatically cross-linked polymers are
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00419
degraded through the pendant reactive groups along the proteolytic degradable
polymeric backbone.32
In addition to chemical structure, the polymer crystallinity, crystal
structures, molecular orientation, melting temperature (Tm), glass transition
temperature (Tg), cross-linking density, external particulates present in the
polymer network, and micro- and nanoscale structure of the scaffold also
influence the degradation rate.33
14.2.6 Biocompatibility
Injectable systems must be compatible with cells, tissues, and bodily fluids in
order to function properly and avoid future complications. Any leachable
compounds and degradation products should not hinder the process of tissue
formation.52 Since injectable materials are often used to deliver cells and
sensitive compounds, the cross-linking process after injection should occur
under physiologically accepted conditions. While designing any injectable
scaffolds, careful consideration towards the toxicity, carcinogenicity, and
chronic inflammatory response induced by the material, cross-linking system,
solvents, and the degradation products should be given special attention.
426 Chapter 14
Chemically cross-linked networks provide higher cross-linking densities to the
polymer network, are more favorable for the sustained release of therapeutics,
and allow for the fabrication of scaffolds with enhanced mechanical properties.
However, the toxicity of the chemical cross-linking agents used may adversely
affect cell behavior and the incorporated bioactive molecules. On the other
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00419
hand, physical gelation of the network may avoid the use of cross-linking
agents, but shows a limited performance in their physical properties. In the next
sections, we will discuss the mechanisms involved in the solidification of
injectable materials.
Figure 14.3 Representative schematic depicting the steps involved in free radical
polymerization network formations.
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428 Chapter 14
14.3.3 Thermally Induced Gelation Systems (TGS)
TGS are widely famous for their use in polymers, which have a unique ability to
undergo sol-to-gel and gel-to-sol phase transitions as a function of temperature.
These polymers are amphiphilic in nature (composed of both hydrophilic and
hydrophobic segments), and display a sol-to-gel transition, which is attributed
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00419
430 Chapter 14
Table 14.2 Biodegradable injectable system properties.
Network
Formation Material Application Reference
polymerization
Chitosan Ligament tissue 93–95
Hyaluronic acid Cartilage tissue 96–98
Poly(ethylene glycol) Bone tissue 40,99–101
based
Poly(L-lactide) based Bone substitute 16,102,103
Poly(vinyl alcohol) Wound tissue 104–105
Poly(propylene Bone tissue 29,31,39,52,
fumarates) 54,106–109
Poly(alkylene maleate Cell delivery 113,110
citrates) Drug delivery
Bone substitute
Chemical cross- Chitosan Cartilage tissue 60
linked systems
Dextran Tissue engineering 63
Protein delivery
Gelatin Cell delivery 61
Hyaluronic acid Protein delivery 62
Poly(ethylene glycol) Bone tissue 50,58
based
Thermally induced Chitosan Cardiac tissue 61,73,111,112
gelation systems Neural tissue
Bone tissue
Poloxamers or Lung tissue 71,113,114
Pluronics Bone tissue
MPEG-PCL 66
MPEG-b-(PCL- 70
ran-PLLA)
PEG-co-poly(a- Drug delivery 67–69
hydroxy acid) Protein delivery
Cell delivery
Self-assembly RGD-based fibers Bone tissue 115
systems
Poly(DL-lactide-co- 80
caprolactone)
Peptide-based hydrogel Bone tissue 116,117
Ion-mediated Alginate Bone tissue 38,118–120
gelation systems
dating as far back as 1892 in the development of plaster of Paris (CaSO4). The
currently available bone cements, which are both injectable and biodegradable,
can be organized into three categories: calcium phosphate cements (CPCs),
bioglass, and bioactive glass cements.121,122
CPCs are widely used as bone substitutes and for augmentation in ortho-
pedic applications due to their close resemblance to the mineral component of
natural bone.90 CPCs are a powder phase of calcium and/or phosphate salts,
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432 Chapter 14
Table 14.3 Cement-based injectable system mechanical properties and
applications.
Compressive
Cement material strength Application Reference
coating materials
Biosorb (b-TCP) 15–150 MPa Bone filler 124
Calcibons 4–7 MPa Bone substitute material 124
ChronOS Inject 7.5 MPa Bone remodeling and cyst 124
treatment
Bone Sourcet 26 MPa Craniotomy cuts and cranial 121,131
defects
Norians SRS 50 MPa Bone fractures 121,132,133
Cementek 20 MPa Bone substitute material 123
45S5sBioglass B500 MPa Bone filler, Middle ear prostheses, 24,134
Periodontal disease
A/W glass ceramic 1080 MPa Artificial vertebrae, Bone fillers, 129,130
Intervertebral discs
Recent strategies involving hyaluronic acid for cell encapsulation for bone
tissue engineering applications have involved modifying hyaluronic acid with
methacrylates and thiols. In vivo studies using acrylated hyaluronic acid
injected into rat calvarial (skull) defects showed that human mesenchymal stem
cells (hMSCs) in the presence of bone morphogenetic factor-2 (BMP-2)
demonstrated the ability to differentiate into specific cells such as endothelial
and osteoblast cells.101,137
434 Chapter 14
Table 14.4 Biodegradable injectable cell delivery vehicles for bone tissue
engineering.
Material Cell Reference
436 Chapter 14
the HAC/gentamicin-treated animals, whereas different stages of chronic
osteomyelitis were found in all control groups.
Although antibiotics have been incorporated into many commercially
available types of cement, the high curing temperatures required and poor
release kinetics of the antibiotic have driven researchers to develop other
delivery vehicles.172 Peng et al.179 have recently developed a novel thermo-
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00419
438 Chapter 14
Table 14.5 (Continued )
Therapeutic Matrix Cell/animal
Carrier material agent type model Reference
degradation profiles, and surface energies, which are all important in control-
ling the biological response to an implanted material. The excellent bio-
compatibility, hemocompatible nature, and tunable mechanical properties of
POC drove Yang et al. to utilize the material primarily for small diameter
vascular grafts226 and medical device coatings.227 Qiu et al. later proposed to
combine POC and HA to create a composite (POC-HA) that would have the
desired characteristics of a bioceramic suitable for orthopedic tissue engineer-
ing.228 Bone screws fabricated from POC-HA composites displayed improved
processability, mechanical properties, and degradation kinetics over previous
biodegradable composites. However, the previous design required harsh pro-
cessing conditions (4120 1C) for polymer network formation rendering them
unable to be used in injectable strategies.
To overcome this limitation, our lab has recently developed a new family of
in situ cross-linkable citric acid-based polymers, which can be cross-linked
through free radical polymerization methods to avoid the use of harsh pro-
cessing conditions required by the previous design. In this system, citric acid,
maleic anhydride or maleic acid, and 1,8-octanediol were reacted together in a
convenient polycondensation reaction to produce a biodegradable elastomer,
poly(alkylene maleate citrates) (PAMC), which could be cross-linked using UV
irradiation or redox systems to form a cross-linked network.229 Maleic anhy-
dride230 and maleic acid110 were both used to introduce a vinyl moiety in order
to allow for network formation under mild conditions. Unlike the previous
citric acid-based designs, this additional cross-linking method allowed for the
preservation of valuable citric acid carboxylic acid and hydroxyl chemistries,
which could be later used to conjugate bioactive molecules into the bulk
material to control cell behavior.231 To ensure that cells and sensitive drugs/
factors could be incorporated and delivered to the injury site, poly(ethylene
glycol) and acrylic acid were introduced into the system to create poly(ethylene
glycol) maleate citrate (PEGMC), which allowed for water solubility and faster
network formation kinetics.13 The encapsulation of NIH 3T3 fibroblasts and
human dermal fibroblasts showed the cytocompatibility of PEGMC and the
controlled drug release using bovine serum albumin demonstrated PEGMC
potential as a suitable cell and drug delivery vehicle.
To widen the application of PEGMC, our lab set out to develop an inject-
able, porous, and strong citric acid based-composite, which could be used as a
delivery vehicle for cells and drugs in bone tissue engineering applications.
PEGMC was combined with various wt.-% of HA to create PEGMC/HA
composites.
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440 Chapter 14
The rationale behind this biomaterial design are:
lities
(2) to create a completely water-soluble material, which was injectable and
provided a suitable environment for the delivery of sensitive cells/
molecules, PEG was chosen a di-functional diol
(3) maleic anhydride introduced a vinyl moiety into the polymer back-
bone, which allowed for network formation using free radical poly-
merization to avoid the harsh processing conditions of previous
designs
(4) to improve the osteointegration capacity and mechanical properties, HA
was incorporated as a composite blend
(5) the pendant carboxylic acid chemistries can react with bicarbonates to
induce gas foaming and create an injectable porous material.
Figure 14.4 (A) Live/dead stain of hFOB 1.19 osteoblasts encapsulated in PEGMC/
HA hydrogel after 7 days. (B) Mineralization in SBF for PEGMC/HA
composite with 40 wt.% HA at 7 days. (C) 10 mm section of PEGMC
scaffold showing the porosity created from a gas foaming technique.
14.9 Conclusions
The context of this chapter aims to discuss the most recent advances in the use
of injectable biodegradable materials for bone tissue engineering. The current
clinical need, design criteria, and material property requirements were
illustrated followed by an overview of the latest material, cellular, and drug
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442 Chapter 14
delivery technologies through the use of injectable systems. Finally, the major
roadblocks pertaining to the field and the future perspectives to address the
current challenges were described. The ability to design injectable systems
shows huge potential for the regeneration of damaged orthopedic tissues
through minimally invasive procedures. While the initial studies are encoura-
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00419
ging and many injectable materials have shown great promise, the regeneration
of mechanically compliant and porous constructs with a vascular supply
remains a challenge. The precisely controlled and cooperative interaction
between the scaffold material, architecture, therapeutics, and cells is imperative
to fully regenerate biologically functional engineered bone. The continued
advancement in material chemistry and a greater understanding of cell–matrix
interactions, metabolic transport, and the cellular events involved in the body’s
natural healing response will be significant steps in the translation of tissue
engineering research into clinical reality.
Acknowledgments
This work was supported in part by an award R21EB009795 from the National
Institute of Biomedical Imaging and Bioengineering (NIBIB), and a National
Science Foundation (NSF) CAREER award 0954109.
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Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/9781849733458-00419
Production of
Polyhydroxybutyrate (PHB)
from Activated Sludge
M. SURESH KUMAR1 AND TAPAN CHAKRABARTI2
1
Solid and Hazardous Waste Management Division, National
Environmental Engineering Research Institute (NEERI),
Nehru Marg, Nagpur 440 020, India; 2 Former Acting Director,
National Environmental Engineering Research Institute (NEERI),
Nehru Marg, Nagpur 440 020, India
15.1 Introduction
Plastics are utilized in almost every manufacturing industry ranging from
automobiles to medicine. The synthetic compounds polyethylene, poly-
vinylchloride and polystyrene are largely used in the synthesis of plastics. These
plastics can be easily moulded into any desired products. Because of these
reasons enormous quantities of plastics have been used and are still in use all
over the world. However, the major problem with plastic is its disposal as
plastics are recalcitrant to microbial degradation. In the recent years, there has
been increasing public concern over the harmful effects of petrochemical-
derived plastic materials in the environment. This has prompted many coun-
tries to start developing biodegradable plastics. Polyhydroxyalkanoates (PHA)
are the polyester of hydroxyalkanoates synthesized by numerous bacteria as
intracellular carbon-storage and energy-storage compounds, and have
mechanical properties similar to polypropylene or polyethylene. Short-chain-
length PHAs such as poly-3-hydroxybutyrate (PHB) have been studied in depth
452
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15.2 Polymers
Polymers are made of two major groups: aliphatic (linear) polymers and aro-
matic (aromatic rings) polymers. Polyhydroxyalkanoates (PHAs) are aliphatic
polymers naturally produced via a microbial process using carbohydrate-based
medium, which acts as carbon-storage and energy-storage materials in bacteria.
They were the first biodegradable polymers to be utilized in plastics. The two
main members of the PHA family are polyhydroxybutyrate (PHB) and poly-
hydroxyvalerate (PHV). Aliphatic polymers such as PHAs, and homopolymers
and copolymers of hydroxybutyric acid and hydroxyvaleric acid, have been
proven to be readily biodegradable. Such polymers are actually synthesized by
microbes, with the polymer accumulating in the microbes’ cells during growth.
The PHB homopolymer is a stiff and rather brittle polymer of high crystallinity,
whose mechanical properties are more or less akin to those of polystyrene,
which, however, is less brittle. PHB copolymers are preferred for general
purposes as the degradation rate of PHB homopolymer is high at its normal
melt processing temperature. PHB and its copolymers with PHV are melt-
processable semi-crystalline thermoplastics made from renewable carbohydrate
feedstocks through fermentation. They represent the first example of a true
biodegradeable thermoplastic produced through the biotechnological route.
No toxic by-products are known to result from PHB or PHV production.
Polyhydroxybutyrate-co-polyhydroxyhexanoates (PHBHs) resins are one of
the newest types of naturally produced biodegradable polymers. The PHBH
resin is derived from carbon sources such as sucrose, fatty acids or molasses via
a fermentation process. These are ‘aliphatic-aliphatic’ copolyesters, as distinct
from ‘aliphatic-aromatic’ copolyesters. Besides being completely biodegrad-
able, they also exhibit barrier properties similar to those exhibited by ethylene
vinyl alcohol. Procter & Gamble Co. researched the blending of these polymers
to obtain the appropriate stiffness or flexibility.
Beside the homopolyester poly-R-3-hydroxybutanoate, consisting of 3-
hydroxybutanoate (3HB) only, two main types of copolyesters can be formed
by different microorganisms.3 The first type of PHAs always contain C3 units
in the polymer backbone; however, the side chains can contain H, and methyl
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454 Chapter 15
(or ethyl) groups if prepared with microorganisms like Ralstonia eutropha.
Propyl to nonyl groups are found in the side chains if the copolyester is
prepared with Pseudomonas oleovorans. In the latter case, branchings,4 double
bonds,5 epoxides6 and aromatic structures7 can be introduced into the side
chain. Furthermore, copolyesters containing o-chloroalkanoates (F, Cl, Br) can
be produced.8–10 In the case of P. oleovorans and other strains from the group
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There are four different pathways for the synthesis of PHAs found to date.
456 Chapter 15
the enzymatic mechanisms of b-ketoacyl-CoA thiolase, acetoacetyl-CoA
reductase, and P(3HB) polymerase have focused on only two of the natural
producers, Zoogloea ramigera and Ralstonia eutropha (formerly known as
Alcaligenes eutrophus).25
Numerous bacteria synthesize and accumulate PHA as carbon- and energy-
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storage materials or as a sink for redundant reducing power under the condi-
tion of limiting nutrients in the presence of excess carbon source. When the
supply of the limiting nutrient is restored, the PHA can be degraded by
intracellular depolymerases and subsequently metabolized as a carbon and
energy source.26,27 Two different types of depolymerase systems had been
recognized, in Rhodospirillum rubrum and B. megaterium. Native granules from
R. rubrum are self hydrolysing, whereas those from B. megaterium are quite
stable, although a soluble extract from R. rubrum was active in the degradation
of native granules from B. megaterium. Purified polymer or denatured granules
did not serve as substrate.28 A soluble PHB depolymerase isolated from B.
megaterium yielded a mixture of dimers and monomer as hydrolysis products,29
whereas the soluble depolymerase from Alcaligenes sp. gave D-(–)-3HB as the
sole product.30 A more detailed study of the B. megaterium system disclosed
that depolymerization required a heat-labile factor associated with the granules
together with three soluble components, namely, a heat-stable protein acti-
vator, PHB depolymerase and a hydrolase.31
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containers, and disposable items such as razors, utensils, diapers and feminine
products.
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63 64,65
glutamic acid in the wastewater, olive oil mill effluents, palm oil mill
effluents,66 soyabean oil67 and agricultural waste.68 However, the polymer
concentration and content obtained were considerably lower than those obtained
using purified carbon substrates. Therefore, there is a need for development of
more efficient fermentation strategies for production of these polymers from a
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15.4.3.2 EBPR
Recently, interest has developed in the use of biological, rather than chemical,
processes for phosphorus removal from wastewater. Sequential anaerobic–
aerobic operation of activated sludge process is applied to achieve enhanced
biological phosphorus removal (EBPR). The simplest system configuration for
EBPR consists of two stages in series, the first one being anaerobic and the
second aerobic. The activated sludge (biomass) is cycled between anaerobic and
aerobic phases, and the influent is supplied under anaerobic conditions.
Operation in this model promotes the accumulation of PAOs.80
During the EBPR anaerobic phase, carbon substrates such as acetic and
propionic acids are taken up to biosynthesize PHV. The energy and reduction
equivalent required for PHA biosynthesis are provided by the degradation of
intracellular saved polyphosphate (poly-P) and glycogen as well as substrate
degradation in the tricarboxylic acid cycle.81–83 The bulk phosphorus con-
centration in the anaerobic period is, therefore, increased with time. In the
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subsequent aerobic phase, where no external carbon source is present, the
internally stored PHA are oxidized and used for cell growth, phosphate uptake
and poly-P accumulation and glycogen synthesis. In the successful EBPR
process, the aerobic phosphate uptake is much higher than the anaerobic
phosphate release, which results in net phosphate removal. The carbon sub-
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strates obviously play an important role in the EBPR process since they are the
raw materials of PHA biosynthesis.
Enhanced biological phosphorus removal (EBPR) is accepted as one of the
most economical and environmentally sustainable processes to remove phos-
phorus (P) from wastewater. It is widely applied for the treatment of domestic
wastes, which have typical P concentrations of between 4 and 12 mg PO4-P
L1.84 However, adoption of EBPR for the treatment of industrial and agri-
cultural wastewaters is less common. These high-strength wastewaters can be
rich in phosphorus, reaching, for example, 125 mg PO4-P L1 in New Zealand
dairy wastewaters.85 There is limited knowledge about the ability of the EBPR
process to deal with such high strength waste streams. The EBPR process relies
on PAOs, which can be encouraged to take up significantly more phosphorus
than is required for cell growth. In order to achieve a population high in PAOs,
an anaerobic contact phase followed by an aerobic contact phase is required.
During the anaerobic phase PAOs convert volatile fatty acids (VFA) into poly-
b-hydroxylalkanoates (PHA). Energy for this process comes mostly from the
use of stored polyphosphates, while the reducing equivalents are provided by
the glycolysis of glycogen86 or alternatively as a product of the TCA cycle.87 As
the polyphosphates are hydrolysed, orthophosphate is released resulting in an
increased phosphate concentration in the bulk liquid. During the aerobic phase
the PAOs use the internally stored PHAs as an energy source to take up
orthophosphate and replenish their polyphosphate reserves. PHA is also used
to drive cell growth and glycogen replenishment. During the aerobic phase the
polyphosphates are accumulated and result in a net reduction of orthophos-
phate from the bulk solution. Phosphorus can then be removed from the system
via wastage of the polyphosphate-rich biomass.
tioned systems for industrial production of PHAs, the feast and famine
approach is the most promising because of high PHA accumulation. This
approach promotes the conversion of the carbon substrate to PHA and not to
glycogen or other intracellular materials.
Mixed cultures or co-culture systems have been recognized to be important
for several fermentation processes. Several studies have claimed the integrity
and effectiveness of the system using mixed cultures. This is due to its complex
nature of dynamics and the difficulty in analysing the dynamics and control of
the system having multiple microorganisms. Unfortunately, the previous
researches only focused on the mixed cultures using two or three well-known
bacteria. The mixed cultures using unknown bacteria have not been paid much
attention due to inconsistent results.
In recent years many studies have been focused on the production of PHAs
by mixed culture when exposed to a transient carbon supply.88,89 Under these
dynamic conditions, the biomass subjected to consecutive periods of external
substrate accessibility (feast period) and unavailability (famine period) gen-
erates a so-called unbalanced growth. During the excess of external carbon
substrate, the growth of biomass and storage of polymer occur simultaneously.
The uptake is mainly driven to PHA storage with low consideration for bio-
mass growth. The microorganisms are able to accumulate substrate as internal
storage products in their cells. Usually these storage products are glycogen,
lipids or PHAs. The final PHA content in the cells will be slightly increased
during transient condition (shifting from feast to famine period).90 After sub-
strate is exhausted, the stored polymer can be used as an energy and carbon
source to enable them to survive the famine period. Hence, the accumulated
PHA will be degraded. Beccari et al.91 reported that the activated sludge is able
to accumulate PHAs up to 50% of cell dry weight.
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components, etc. However, if the supply of oxygen is adequately controlled, the
assimilative activity will be suppressed while letting the microorganism accu-
mulate PHA. By using these conditions, PHA accumulators are selected
regardless of the ability of microorganisms to accumulate poly-P or glycogen,
and the selected PHA accumulators will have a lower tendency to accumulate
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Microorganisms which are able to quickly store available substrate and con-
sume the storage to achieve a more balanced growth have a strong competitive
advantage over organisms without the capacity of substrate storage.99 Acti-
vated sludge accumulates PHA to around 20% of dry weight under anaerobic
conditions. The PHA content of activated sludge can be increased to 62% in a
microaerophilic–aerobic sludge process.94,100 When compared with a pure
culture (more than 88% of cell dry weight),39 the merits of PHA production in
open mixed culture would be an enhanced economy, a simpler process control,
no requirement of monoseptic processing, and an improved use of wastes. A
considerable effort has gone in producing PHA using mixed culture.
Several methods have been used as a recovery process for PHA. These
methods include solvent extraction, sodium hypochlorite digestion and enzy-
matic digestion. Details of each method as well as their advantages and dis-
advantages will be discussed and summarized here. In most cases, bacterial
biomass is separated from substrate medium by centrifugation, filtration or
flocculation. Then, the biomass is freeze dried (lyophilized). Basically, mild
polar compounds, e.g. acetone and alcohols, solubilize non-PHA cellular
materials whereas PHA granules remain intact. Non-PHA cellular materials
are nucleic acids, lipids, phospholipids, peptidoglycan and proteinaceous
materials. On the other hands, chloroform and other chlorinated hydrocarbons
solubilize all PHAs. Therefore, both types of solvents are usually applied
during recovery process. Finally, evaporation or precipitation with acetone or
alcohol can be used to separate the dissolved polymer from the solvent. PHAs
are soluble in solvents, such as chloroform, methylene chloride or 1,2-
dicholoroethane. These three solvents can be used to extract PHA from
bacterial biomass. In addition, other solvents were also reported to be used to
extract PHA, e.g. ethylene carbonate, 1,2-propylene carbonate, mixtures of
1,1,2-trichloroethane with water and mixtures of chloroform with methanol,
ethanol, acetone or hexane.
Doi101 described a chloroform extraction method. PHA is extracted with hot
chloroform in a Soxhlet apparatus for over 1 hour. Then, PHA extracted is
separated from lipids by precipitating with diethyl ether, hexane, methanol or
ethanol. Finally, PHA is redissolved in chloroform and further purified by
precipitation with hexane. Ramsay et al.102 examined the recovery of PHA
from three different chlorinated solvents (chloroform, methylene chloride and
1,2-dichloroethane). They obtained the best recovery and purity when biomass
was pre-treated with acetone. The optimum digestion time for all three solvents
were 15 minutes. Further digestion resulted in a reduction in the molecular
weight (MW) of PHA. The degree of recovery when the biomass was pre-
treated with acetone were 70, 24 and 66% when refluxed for 15 minutes with
chloroform, methylene chloride and 1,2-dichloroethane, respectively. The level
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466 Chapter 15
of purity of these three solvents under these optimum conditions were 96, 95
and 93%, respectively. Temperatures used with these three solvents were 61, 40
and 83 1C, respectively. The authors emphasized that extraction conditions
have a great impact on the degradation of PHA during the recovery process.
Sodium hypochorite solubilizes non-PHA cellular materials and leaves PHA
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used; the resulting solution is filtered to remove debris and concentrated, and
the polymer is precipitated with methanol or ethanol, leaving low-molecular-
weight lipids in solution. Longer-side-chain PHAs show a less restricted
solubility than PHB and are, for example, soluble in acetone. However, the
large-scale use of solvents is not economic commercially, and other strategies
have been adopted.
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results in a high recovery cost. This is mainly due to the use of large amounts of
digesting agents for breaking the cell walls and to the increased cost of waste
disposal.
15.8 Conclusions
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Subject Index
Page references to figures and tables are shown in italics.