screening and study on microorganisms degrading biopolymers in vietnam

Table of contents
INTRODUCTION 1
CHAPTER 1: LITERATURE REVIEW 4
1.1. PLASTIC WASTE POLLUTION 4
1.1.1. Plastic waste pollution in the world 4
1.1.2. Plastic waste pollution in Vietnam 5
1.1.3. Treatment of plastic waste 6
1.1.3.1. Landfill 6
1.1.3.2. Recycling 6
1.1.3.3. Incineration 7
1.2. BIODEGRADABLE PLASTICS 8
1.2.1. Biodegradable plastics 8
1.2.1.1. Poly(Lactic Acid) (PLA) 11
1.2.1.2. Poly(3-Hydroxybutyrate) (PHB) 12
1.2.1.3. Poly(ε-Caprolactone) (PCL) 13
1.2.2. Applications 13
1.2.2.1. Medicine and pharmacy 14
1.2.2.2. Packaging 15
1.2.2.3. Agriculture 15
1.2.2.4. Others fields 16
1.3. THE DEGRADATION OF BIOPOLYMERS 17
1.4. MICROORGANISMS DEGRADING BIODEGRADABLE POLYMERS . 19
1.4.1. Microorganisms degrading PLA 19
1.4.2. Microorganisms degrading PHB 22
1.4.3. Microorganisms degrading PCL 23
CHAPTER 2: MATERIALS AND METHODS 25
2.1. MATERIALS 25
2.2. CHEMICALS 25
2.3. EQUIPMENTS 26
2.4. METHODS 26
2.4.1. Isolation of biopolymer-degrading microorganisms 26

2.4.2. Screening biopolymer-degrading microorganisms 27
2.4.3. Identification of biopolymers-degrading strains 27
2.4.3.1. Gram staining method 27
2.4.3.2. Observation under scanning electron microscopy (SEM) 28
2.4.3.3. Extraction of genomic DNA from bacteria 29
2.4.3.4. Amplification of the 16S rDNA PCR reaction 29
2.4.3.5. Agarose gel electrophoresis 30
2.4.3.6. Sequencing 30
2.4.3.7. Effect of culture conditions 31
2.4.3.8. Utilization of of sugars 31
2.4.3.9. Activity of some extracellular enzymes 31
2.4.4. Study degradation of biodegradable polymers by isolated strains 32
2.4.4.1. Growth experiment in the PLA, PHB or PCL containing media 32
2.4.4.2. Measurement of the PLA, PHB or PCL residual weight 32
2.4.4.3. Determination of TOC in culture broth 32
2.4.4.4. Degradation experiment with biopolymer film 34
2.4.4.5. Statistical analysis 34
CHAPTER 3: RESULTS AND DISCUSSION 35
3.1. ISOLATION AND SCREENING PLA, PHB, PCL-DEGRADING ORGANISMS 35
3.2. PLA-DEGRADING MICROORGANISMS 37
3.2.1. Identification of strains G5 and Cz1 37
3.2.1.1 Morphology of strain G5 and Cz1 38
3.2.1.2. 16S rDNA sequencing of strain G5 39
3.2.1.3. Biochemical and physiological characteristics of strains G5 and Cz1 41
3.2.2. PLA degradation by S. thermoflavus G5 and P. citrinium Cz1 44
3.3. PHB-DEGRADING MICROORGANISM 47
3.3.1. Identification of strain B2 47
3.3.1.1. Morphology of strain B2 47
3.3.1.2. Sequencing 16S rDNA gene of strain B2 48
3.3.1.3. Biochemical and physiological characteristics of strain B2 49

3.3.2. PHB degradation by B. gelatini B2 52
3.4. PCL-DEGRADING MICROORGANISM 54
3.4.1. Identification of strain B1 55
3.4.1.1. Morphology of strain B1 55
3.4.1.2. Sequencing 16S rDNA gene of strain B1 56
3.4.1.3. Biochemical and physiological characteristics of strain B1 57
3.4.2. PCL degradation by Br. agri B1 60
3.5. DEGRADATION OF POLYMERS BY ISOLATED STRAINS 61
CONCLUSIONS 64
FURTHER STUDY 65
REFERENCES 66
WEB REFERENCES
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INTRODUCTION
1. The urgency of the subject:
In recent years, environmental pollution is a major concern of many
countries around the world. Pollution caused by plastic waste seems to increase.
Use of polymer products that derived from biological sources replaced synthetic
plastic products is a new direction in reducing environmental pollution. Bio-plastics
aggregated from many different types of polyesters as poly(lactic acid) (PLA),
poly(3-hydroxyrate (PHB), poly(ε-caprolactone) currently have attracted much
attention because of their potential application in many fields such as: in packaging,
agriculture, medicine, biodegradable plastics and other areas. Biodegradable
polymers that were capableof degradation by both microorganisms and enzymes are
currently considerable as the sustainable recycling method for polymers.
Microorganism is one of the factors affecting the degradation of bio-polymers.
Polymer-degrading microorganisms could be found in different environments such as

soil, sea, water, compost, activated sludge… [55]. In this connection, scientists are
now focusing on isolation and selection of microorganisms that are able to increase
polymer degradation. Recently, various investigations of microbial degradation of
polymers have been published [23], [56], [63].
In Vietnam, environmental pollution caused by plastic waste is an alarming
issue. The increasing use of bio-polymer products in day life promotes both
research and application of polymer-degrading microbes to reduce environmental
pollution. Thus, isolation and selection of strains that can degrade bio-polymers
were initially carried out. However, information about microorganisms degrading
biopolymers and application is still very limited [32], [33], [34]. The topic:
"Screening and study on microorganisms degrading biopolymers in Vietnam" was
therfore inplemented in order to contribute to solving the existing plastic pollution
problem in Vietnam.
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2. The aim of the study
- Screen microorganisms that are capable of degrading PLA, PHB and PCL.
- Classify the isolated strains and study optimal conditions for the growth of
these strains.
- Biopolymer-degradation activity of the isolated strains.
3. Content of the study
Screening microorganisms capable of degrading PLA, PHB and PCL by
clear-zone methods and by measuring the growth of microorganisms in the medium
supplemented with polymers.
Identification of isolated strains based on the phenotypic, biochemical and
physiological characteristics together with 16S rDNA sequencing.
Study the polymer degradation of the isolated strains by measuring the total
organic carbon (TOC) and measuring the loss of polymer residual in the culture
broth.

4. Practical applicability
In the future, we intend to apply these isolated strains in degradation of
biopolymers in the compost.
5. Contribution of the study
It is the first time strains capable of degrading PLA, PHB and PCL were
screened in Vietnam.
Based on the phenotypic, biochemical and physiological characteristics
together with 16S rDNA sequencing, strains G5 and Cz1 that were capable of
degrading PLA were indentified as Streptomyces thermoflavus and Penicillium
citrinum, respectively. Thus, this is the first publication about strains belonging to
genera Streptomyces and Penicillium that were capable of degrading PLA.
It is also the first time thermophilic members of genus Bacillus that were
capable of degrading PHB and PCL were published. Based on the phenotypic,
biochemical and physiological characteristics together with 16S rDNA sequencing,
these strains were identified as Bacillus gelatini and Brevibacillus agri.
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6. Distribution of thesis
Thesis contained 85 pages, 5 tables, 35 figures, and 74 references.
Beside introduction, conclusions, futher study, reference and appendix, thesis
concluded 3 chapters:
Chapter 1: Literature review
Chapter 2: Materials and methods
Chapter 3: Results and discussion


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Chapter 1: LITERATURE REVIEW
1.1. PLASTIC WASTE POLLUTION
1.1.1. Plastic waste pollution in the world
Environmental pollution is one of the biggest subjects in nowadays. There
are many kinds of pollution, such as air, water, noise, soil pollution…, among them,
solid waste pollution is the most pressing issues. The quantity of solid waste is
greatly increasing due to the increase of population, development activities, and
changes in life style.
Plastic waste of all kinds presents a significant and costly form of pollution.
In the United Kingdom, plastics made up around 7% of the average household
dustbin. The amount of plastic waste generated annually in the United Kingdom
was estimated to be nearly 3 million tons in 2002. It was estimated that 56% of all
plastics waste is used packaging, three-quarters of which is from households,
however only 7% of total plastic waste has been recycled [67]. This situation was
similar in the United State, India and many other countries [68], [71]. In the world,
annually 100 million tons of plastics has been used, thus causing a significant
problem for the environment [49].
The plastic pollution causes a range of environmental impacts. Plastic
production requires significant amount of resources, primarily fossil fuels, both as a
raw material and energy source for the manufacturing process. According to World
Watch Institute (United State), it takes 430,000 gallons of oil to produce 100 million
plastic bags. In the world, 4% of the annual oil production is used as a feedstock for
plastic production and additionally 3-4% as energy source for manufacturing
process [67], [69].
Plastic production also involves the use of potentially harmful chemicals,
which are added as stabilizers or colorants. Many of these have not undergone
environmental risk assessment and their impact on human health and the
environment is currently uncertain. In developing countries, plastic bags are used to
carry food by citizens - as they are cheap, convenient and air tight. Liquid, spicy or

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fatty food items are packed in colored plastic bags, and carcinogens are likely to be
generated during chemical reactions taken place in the bags, due to temperature
variations. Colored nylon bags used to contain food, can infect contents with metals
like lead and cadmium, which harm human’s brains and lungs [38], [67].
The disposal of plastic products also contributes significantly to their
environmental impact. The presence of plastic in our environment is killing many
animal species [69]. According to Greenpeace, more than 1 million birds and
100,000 marine mammals are estimated to perish each year by either eating or
becoming trapped in plastic waste. Sea turtles, whales and dolphins are among sea
animals being directly affected by plastic waste products, often mistaking plastic
bags for food, causing slow and painful deaths to these animals over a prolonged
period of time [69].
1.1.2. Plastic waste pollution in Vietnam
Pollution caused by solid waste is an alarming issue in Vietnam. Untreated
waste now affects the environment, land, water as well as people's health.
According to a report, the quantity of solid waste in all cities increased significantly,
from 1478 tons in 2000 at Lamson landfill (Hanoi) to 2540 tons in 2004. At this
time, the total amount of solid waste released in Vietnam is around 28 million tons
per year. In particular, in 2004, the amount of solid waste was 15.5 million tons. It
is predicted that the amount in 2015, 2020, and 2025 would be approximately 43.6,
67.6, and 91.6 million tons, respectively [70].
Plastic waste was a significant portion of the total solid waste. It is estimated
that plastic waste occupied approximately 10% of municipal solid waste [70]. A
large portion of plastic waste pollution came from plastic bags. Because buyers
found them comfortable to use while sellers used them as an effective advertising
tool and companies enjoy diversifying the design of their plastic bags to attract
more customers, thus the bags themselves were used more and more popular.

According to Ministry of Natural Resources and Environment of Vietnam, each
household uses five plastic bags per day, and that amounts to 90 million bags in the
entire country. Among them, only 3-4% of plastic in Vietnam is recycled, while the
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rest is dumped or buried into soil. This situation leads to severe pollution and affect
to the citizen life.
1.1.3. Treatment of plastic waste
There are several ways to treat the pollution of the plastic waste in the world
and in Vietnam.
1.1.3.1. Landfill
A landfill is a site for the disposal of waste materials by burial and is the
oldest form of waste treatment. Historically, landfills have been the most common
methods of organized waste disposal and remain so in many places around the
world. Landfills may include internal waste disposal sites (where a producer of
waste carries out their own waste disposal at the place of production) as well as sites
used by many producers. Many landfills are also used for other waste management
purposes, such as the temporary storage, consolidation and transfer, or processing of
waste material (sorting, treatment, or recycling).
In the past, plastic waste was not separated from waste. It was dumped at the
landfill and remained a long time after, so a large number of adverse impacts may
occur. Because most plastics are non-degradable, they take a long time to break
down, possibly up to hundreds of years [73]. Over time they go through a process of
light degradation and break down into smaller pieces that cannot be converted by
any known organism and as such remain as plastic in landfills, rivers and oceans.
With more and more plastic products, particularly plastics packaging, the landfill
space required for plastics waste is a growing concern. Thus, plastic bags can choke
the earth, they are making soil unfertile, contaminate ground and water through
leaching of toxic substances. Recently, modern landfills in industrialized countries

are operated with controls to attempt manage the problems, the plastic waste is not
dumped to the landfill, instead of, it is isolated from rubbish and treated in special
way.
1.1.3.2. Recycling
Recycling used to prevent waste of potentially useful materials. Recyclable
materials include many kinds of glass, paper, metal, plastic, textiles, and electronics.

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It reduces the consumption of fresh raw materials, reduces energy usage, reduces air
pollution (from incineration) and water pollution (from landfilling) by reducing the
need for "conventional" waste disposal, and lower greenhouse gas emissions as
compared to virgin production.
The United States Environmental Protection Agency (EPA) has concluded
that recycling reduced the country's carbon emissions by a net of 49 million metric
tons in 2005 [72]. In the United Kingdom, the Waste and Resources Action
Programme stated that Great Britain's recycling reduced CO
2
emissions by 10-15
million tons a year [72]. However, this work was often difficult or too expensive
(compared with producing the same product from raw materials or other sources),
therfore only a small quantity of plastic waste has been recycled.
1.1.3.3. Incineration

Fig 1. Incinerator in Hinwil, Switzerland
[73].
Fig 2. Incinerator in Nam Dinh province,
Vietnam [74].
Incineration is a waste treatment technology that involves the combustion of

organic materials and substances. In developed countries, incineration of waste
materials converts the waste into incinerator bottom ash, flue gases, particulates,
and heat, which can in turn be used to generate electric power. The flue gases are
cleaned before they are dispersed in the atmosphere. In developing countries, this
solution is only used as burn materials.
Incinerators reduce the mass of the original waste by 80–85 % and the
volume (already compressed somewhat in garbage trucks) by 95-96 %, depending
upon composition and degree of recovery of materials such as metals from the ash
for recycling [73].

Thus, it reduces the necessary volume for disposal significantly.
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However, burn of the plastic waste significantly harms human health and the
environment by emitting toxic chemicals such as sulfur dioxide and even dioxin
[38]. It is the reason why, incinerators have been used with the limitation in many
countries.
1.2. BIODEGRADABLE PLASTICS
With the advances in technology and the increase in the global population,
plastic materials have found wide applications in every aspect of the life and
industries. However, most conventional plastics such as polyethylene,
polypropylene, polystyrene, poly(vinyl chloride) and poly(ethylene terephthalate),
are non-biodegradable, and their increasing accumulation in the environment has
been a threat to the planet. To overcome these problems, the strategy involved
production of plastics with high degree of degradability is developed [55].
The American Society for Testing of Materials (ASTM) and the International
Standards Organization (ISO) define degradable plastics as those which undergo a
significant change in chemical structure under specific environmental conditions.
These changes result in a loss of physical and mechanical properties, as measured

by standard methods. Biodegradable plastics undergo degradation from the action of
naturally occurring microorganisms such as bacteria, fungi, and algae. Plastics may
also be designated as photodegradable, oxidatively degradable, hydrolytically
degradable, or those which may be composted [24].
The development of innovative biopolymer materials has been underway for
a number of years, and continues to be an area of interest for many scientists. In
2001, the worldwide consumption of biodegradable polymers has increased from 14
million kg in 1996 to an estimated 68 million kg [14]. Recently, in 2009, demand
for biodegradable polymers in North America, Europe and Asia accounted for most
of the global consumption (Fig 3). Europe has been the biggest market of
biodegradable polymers with the consumption of half of the consumption in the
world. North America also occupied a significant consumption of biodegradable
polymers with about 25% of total consumption. In Asia, the consumption of
biodegradable polymers was highest in Japan with around 6% amount of
biodegradable polymers in the world [68].
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Fig 3. World consumption of biodegradable polymers in 2009 [68].
Despite the economic crisis, which hit the chemical and plastic industry, the
market of biodegradable polymers did grow in 2009 in almost all regions. In
Europe, the largest global market, growth was in the range of 5–10% (depending on
products and applications), compared with 2008. Total consumption of
biodegradable polymers in these three regions is forecast to grow at an average
annual rate of nearly 13% over the five-year period from 2009 to 2014. The food
packaging, dishes and cutlery market is the single largest end use and will be the
major growth driver in the future [28].
1.2.1. Biodegradable plastics
Bio-plastics consist of either biodegradable plastics i.e., plastics produced

from fossil materials) or bio-based plastics (i.e., plastics synthesized from biomass
or renewable resources) [55]. The inter-relationship between biodegradable plastics
and bio-based plastics is shown in Fig 4. Poly(ε-caprolactone) (PCL), and
poly(butylene succinate) (PBS) are petroleum based, but they can be degraded by
microorganisms. On the other hand, poly(3-hydroxybutyrate) (PHB), poly(lactide)
(PLA) and starch blends are produced from biomass or renewable resources, and
thus are both biodegradable and bio-based plastic. Despite the fact that polyethylene
(PE) and Nylon 11 (NY11) can be produced from biomass or renewable resources,
they are non-biodegradable. Acetyl cellulose (AcC) is either biodegradable or non-
biodegradable, depending on the degree of acetylation. AcC’s with a low
acetylation can be degraded, while those with high substitution ratios are non-
biodegradable [55].
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Biodegradable plastics are seen by many as a promising solution to reduce
pollution problem because they are environmentally-friendly. They can be derived
from renewable feedstocks, thereby reducing green-house gas emissions. For
instance, polyhydroxyalkanoates (PHA) and lactic acid (raw materials for PLA) can
be produced by fermentative biotechnological processes using agricultural products
and microorganisms. Biodegradable plastics offer a lot of advantages such as
increase soil fertility, low accumulation of bulky plastic materials in the
environment (which invariably will minimize injuries to wild animals), and
reduction in the cost of waste management. Furthermore, biodegradable plastics can
be recycled to useful metabolites (monomers and oligomers) by microorganisms
and enzymes [55].

Fig 4. Bio-plastics comprise biodegradable plastics and bio-based plastics [55]
Among many biodegradable polymers PLA, PHB, PCL seem to be of the
most attention. Because of chemical and physical feature that are suitable for

application in many fields and their degradable ability, in this paper we only
concentrate to present properties and the degradation of these polymers.
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1.2.1.1. Poly(Lactic Acid) (PLA)
O
O
O
O
O
n
HO
HO

Recently, PLA has come to be considered as a potential polymeric material
due to its various advantages. For example, it is regarded as renewable plastic since
its raw material, lactic acid, can be produced by fermentation of biomass on
feedstock including sucrose and corn and tapioca starches. It is expected that PLA
produced by fermentative processes will replace many conventional plastics
produced from petrochemicals. Industrial lactic acid-producing microorganisms
mainly produce L-lactic acid at high concentration of over 100 g/l, with low
production of D-lactic acid. At high concentration and purity, only a small amount
of energy is required for recovery [30], [56].
PLA is a biodegradable and biocompatible thermoplastic. It can also be
synthesized either by condensation polymerization of lactic acid or by ring-opening
polymerization of an intermediate called lactide (a cyclic dimer of lactic acid). This
polymer exists in the form of three stereoisomers: poly(L-lactide) (PLLA), poly(D-
lactide) (PDLA) and poly(DL-lactide) (PDLLA). A semi-crystalline polymer
(PLLA) (crystallinity about 37%) is obtained from L-lactide whereas poly(DL-

lactide) is an amorphous polymer. Their mechanical properties and their
degradation times are different. PLLA is a hard, transparent polymer with an
elongation at break of 85%-105% and a tensile strength of 45 - 70 MPa. It has a
melting point of 170-180°C and a glass transition temperature of 53°C [27].
PDLLA has no melting point and a Tg around 55°C. It shows much lower tensile
strength [66]. Thus, when the optical purity of PLA is low, such as in the case of
PDLLA, the Tm decreases and most its conventional and desirable plastic
properties are lost.
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1.2.1.2. Poly(3-Hydroxybutyrate) (PHB)

PHB ([-O(CH
3
)CHCH
2
CO-]n) is a natural polymer produced by many
bacteria as a means to store carbon and energy. This product has the same
thermoplastic and water resistant qualities as the synthetic plastics. On the other
hand, it is naturally degradable, environmentally friendly substitutes for synthetic
plastics [24]. PHB has attracted scientific and commercial interest worldwide
because it can be synthesized from renewable low-cost feed-stocks and the
polymerizations are operated under mild process conditions with minimal
environmental impact. Furthermore, it can be biodegraded in both aerobic and
anaerobic environments, without forming any toxic products [26], [55].
PHB is 100% stereospecific, with all of the asymmetric carbon atoms in D(-)
configuration. It in vivo is an amorphous polymer that becomes partially crystalline
after release from accumulating cells, after cell lysis. The crystallinity ranges from
55-80% and is relatively stiff. Crystalline PHB is referred to as “denatured”, in

contrast to the amorphous “native form in vivo”[11], [26], [42].
Since 1925, this polyester is produced biotechnologically and was attentively
studied as biodegradable polyester. PHB has methyl groups attached to the main
chain in a single conformation. PHB can have average molecular weight of 0.1 - 3
MDa, although for processing the molecular weights are usually in the range of 200
to 800 kDa. PHB is highly crystalline with crystallinity above 50%. Its melting
temperature is 180°C. The pure homo-polymer is a brittle material. Its glass
transition temperature is approximately 55°C. PHB is susceptible to thermal
degradation at temperatures of the melting point [5], [66].
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1.2.1.3. Poly(ε-Caprolactone) (PCL)




Poly(ε-caprolactone), PCL, is a thermoplastic biodegradable polyester
synthesized by chemical conversion of crude oil, followed by ring-opening
polymerization of ε-caprolactone in presence of catalyst [14], [66]. ε-caprolactone is
a relatively cheap cyclic monomer. PCL has good water, oil, solvent, and chlorine
resistance, a low melting point, and low viscosity. PCL is soluble in a wide range of
solvents. Its glass transition temperature is low, around -60°C, and its melting point
is 60 – 65°C [66]. PCL is a semi-rigid material at room temperature, has a modulus
in the range of low-density polyethylene and high-density polyethylene, a low
tensile strength of 23 MPa and a high elongation to break (more than 70%). Thanks
to its low Tg, PCL is often used as a compatibilizer or as a soft block in
polyurethane formulations. This polymer is also used as an additive for resins to
improve their processing characteristics and their end use properties. Being
compatible with a range of other materials, PCL can be mixed with starch to lower

its cost and increase biodegradability or it can be added as polymeric plasticizer to
PVC.
1.2.2. Applications
Biodegradable polymers used as biomaterials have been recently reviewed.
To be used as biomaterials, biodegradable polymers should have three important
properties: biocompatibility, bioabsorbility and mechanical resistance. The use of
enzymatically degradable natural polymers, as proteins or polysaccharides, in
biomedical applications began thousands of years ago whereas the application of
synthetic biodegradable polymers dates back some fifty years [66]. From then on,
synthetic biodegradable polymers applied to replace petroleum-based polymers
were used widely. The three main sectors where biodegradable polymers have been
introduced include medicine, packaging and agriculture. As biopolymers have a low
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solubility in water and a very important water uptake, they could be used as
absorbent materials in horticulture, healthcare and agricultural applications.
Packaging waste has caused increasing environmental concerns. Thus, the
development of biodegradable packaging materials has received increasing attention
[24], [66]. Other applications of biodegradable polymers were also developed.
1.2.2.1. Medicine and pharmacy
Current applications of biodegradable polymers include surgical implants in
vascular or orthopaedic surgery and plain membranes. Biodegradable polyesters are
widely employed as porous structure in tissue engineering because they typically
have good strength and an adjustable degradation speed. Biodegradable polymers
are also used as implantable matrices for the controlled release of drugs inside the
body or as absorbable sutures [66].
PLA can be considered as the first biodegradable polymers used in
biomedical applications. Due to their good mechanical properties, PLLA have been
used as bone internal fixation devices. It also has excellent fiber forming properties

and thus PLLA was used to replace ligament and non-degradable fibers. As has
lower mechanical properties and faster degradation rate than PLLA, it is often used
in drug delivery systems and scaffolding matrices for tissue engineering [40], [19].
PHB is soluble in a wide range of solvents and can be process in various
shapes. It is used in applications where electrical simulation is applied. PHB has the
advantageous property of being degraded in D-3-hydroxybutyrate, a natural
constituent of human blood. As a consequence, PHB is suitable for biomedical
applications for example it is used in drug carriers and tissue engineering scaffolds
[26].
PCL and their copolymers are also utilized as biomedical materials. PCL is
used as a matrix in controlled release systems for drugs, especially those with
longer working lifetimes. PCL has a good biocompatibility and is used as scaffolds
for tissue engineering. PBS is a promising substance for bone and cartilage repair.
Its processability is better than that of PLA [66].
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1.2.2.2. Packaging
In everyday life, packaging is another important area where biodegradable
polymers are used. In order to reduce the volume of waste, biodegradable polymers
are often used. Besides their biodegradability, biopolymers have other
characteristics as air permeability, low temperature sealability and so on.
Biodegradable polymers used in packaging require different physical
characteristics, depending on the product to be packaged and the store conditions
[24], [40], [66].
Due to its availability and its low price compared to other biodegradable
polyesters, PLA is used for lawn waste bags [19]. In addition, PLA has a medium
permeability level to water vapor and oxygen. It is thus developed in packaging
applications such as cups, bottles, films. PCL finds applications in environment e.g.
soft compostable packaging. PHB has been used in small disposable products and in

packing materials [26], [66].
1.2.2.3. Agriculture
For this application, the most important property of biodegradable polymers
is in fact their biodegradability. Plastic films were first introduced for greenhouse
coverings fumigation and mulching in the 1930s. Young plants are susceptible to
frost and must be covered. The main actions of biodegradable cover films are to
conserve the moisture, to increase soil temperature and to reduce weeds in order to
improve the rate of growth in plants. At the end of the season, the film can be left
into the soil, where it is biodegraded. Another application bases on the production
of bands of sowing. It is bands which contain seeds regularly distributed as well as
nutriments [24], [66].
Biodegradable polymers can be used for the controlled release of agricultural
chemicals. The active agent can be dissolved, dispersed or encapsulated by the
polymer matrix or coating, or is a part of the macromolecular backbone or pendent
side chain. The agricultural chemicals concerned are pesticides and nutrients,
fertilizer, pheromones to repel insects. The natural polymers used in controlled
release systems are typically starch, cellulose, chitin, aliginic acid and lignin [66].
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Agricultural applications for biopolymers are not limited to film covers. In
horticulture threads, clips, staples, bags of fertilizer, envelopes of ensilage and trays
with seeds are applications mentioned for biopolymers. Containers such as
biodegradable plant pots and disposable composting containers and bags are other
agricultural applications. The pots are seeded directly in the soil, and break down as
the plant begins to grow. In marine agriculture, biopolymers are used to make ropes
and fishing nets. They are also used as support for the marine cultures [24].
1.2.2.4. Others fields
Biopolymers are also used in shape specific applications such as in the
automotive, electronics or construction sectors.

Automotive: The automotive sector aims to prepare lighter cars by use of
bioplastics and biocomposites. Natural fibers can replace glass fibers as
reinforcement materials in plastic car parts. We await the development of the bio-
composite materials. For example the PLA is mixed with fibers of kenaf for replace
the panels of car doors and dashboards (Toyota Internet site). Starch-based
polymers are used as additive in the manufacturing of tires (Fig 5). It reduces the
resistance to the movement and the consumption of fuel and in fine greenhouse gas
emissions [66].
Electronics: PLA and kenaf are used as composite in electronics applications.
Compact disks based on PLA are also launched on the market by the Pioneer and
Sanyo groups. Fujitsu Company has launched a computer case made of PLA [65],
[66].
Construction: PLA fiber is used for the padding and the paving stones of
carpet. Its inflammability, lower than that of the synthetic fibers, offers more
security. The fiber is resistant to UV radiation [66].
Sports and leisure: PLA fiber is used for sports clothes. It combines the
comfort of the natural fibers and the resistance of synthetic fibers [66].
There are a lot of other applications such as: combs, pens (Begreen® from
Pilot Pen or Green Pen® from Yokozuna) (Fig 5), and mouse pads made of
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biodegradable polymers have also been invented, mostly for use as marketing tools.
PLA (semi-synthetic polymers) is used for compostable food [66].

Fig 5. Application of polymers in different fields.
1.3. THE DEGRADATION OF BIOPOLYMERS
Just as important as the way in which a material is formed is the way in
which it is degraded. Biodegradation of materials occurs in various steps. Initially,
the digestible macromolecules, which join to form a chain, experience a direct

enzymatic scission. This is followed by metabolism of the split portions, leading to
a progressive enzymatic dissimilation of the macromolecule from the chain ends.
Oxidative cleavage of the macromolecules may occur instead, leading to
metabolization of the fragments. Either way, eventually the chain fragments become
short enough to be converted by microorganisms [24], [45].
An approach to degradation of biopolymers involves growing
microorganisms for the specific purpose of digesting polymer materials. This is a
more intensive process that ultimately costs more, and circumvents the use of
renewable resources as biopolymer feed-stocks. The microorganisms under
consideration are designed to target and breakdown petroleum based plastics.
Although this method reduces the volume of waste, it does not aid in the
preservation of non-renewable resources [24].
Photodegradable polymers undergo degradation from the action of sunlight.
In many cases, polymers are attacked photo-chemically, and broken down to small
pieces. Further microbial degradation must then occur for true biodegradation to be
achieved. Proposed approaches for further developing photodegradable biopolymers
includes incorporating additives that accelerate photochemical reactions (e.g.
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benzophenone), modifying the composition of the polymers to include more UV
absorbing groups (e.g. carbonyl), and synthesizing new polymers with light
sensitive groups. An application for biopolymers which experience both microbial
and photo-degradation is in the use of disposable mulches and crop frost covers
[24].
Some biodegradable polymer materials experience a rapid dissolution when
exposed to particular (chemically based) aqueous solutions. Similar to many
photodegradable plastics, full biodegradation of them occurs later, through
microbial digestion. The appropriate microorganisms are conveniently found in
wastewater treatment plants [24].

 Factors affecting the biodegradability of plastics
The properties of plastics are associated with their biodegradability. Both the
chemical and physical properties of plastics influence the mechanism of
biodegradation. The surface conditions (surface area, hydrophilic, and hydrophobic
properties), the first order structures (chemical structure, molecular weight and
molecular weight distribution) and the high order structures (glass transition
temperature, melting temperature, modulus of elasticity, crystallinity and crystal
structure) of polymers play important roles in the biodegradation processes [55].
In general, polyesters with side chains are less assimilated than those without
side chains. The molecular weight is also important for the biodegradability because
it determines many physical properties of the polymer. Increasing the molecular
weight of the polymer decreased its degradability. PCL with higher molecular
weight (Mw > 4,000) was degraded slowly by Rhizopus delemar lipase (endo-
cleavage type) than that with low Mw [55].
Moreover, the morphology of polymers greatly affects their rates of
biodegradation. The degree of crystallinity is a crucial factor affecting
biodegradability, since enzymes mainly attack the amorphous domains of a
polymer. The molecules in the amorphous region are loosely packed, and thus make
it more susceptible to degradation. The crystalline part of the polymers is more
resistant than the amorphous region. The rate of degradation of PLA decreases with
an increase in crystallinity of the polymer [16], [55].
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The melting temperature of polyesters has a strong effect on the enzymatic
degradation of polymers. The higher the Tm is, the lower the biodegradation of the
polymer is [55].
1.4. MICROORGANISMS DEGRADING BIODEGRADABLE POLYMERS
Biodiversity and occurrence of polymer-degrading microorganisms vary
depending on the environment, such as soil, sea, compost, activated sludge, etc. It is

necessary to investigate the distribution and population of polymer-degrading
microorganisms in various ecosystems. Generally, the adherence of microorganisms
on the surface of plastics followed by the colonization of the exposed surface is the
major mechanisms involved in the microbial degradation of plastics. The enzymatic
degradation of plastics by hydrolysis is a two-step process: first, the enzyme binds
to the polymer substrate then subsequently catalyzes a hydrolytic cleavage.
Polymers are degraded into low molecular weight oligomers, dimers and monomers
and finally mineralized to CO
2
and H
2
O [55].
The ecological and taxonomic studies on the abundance and diversity of
polymer-degrading microorganisms in the different environment are necessary
because they are responsible for the degradation of plastic materials. Polymers are
degraded in the soil by the action of a wide variety of microorganisms. The plate
count and the clear zone methods using emulsified polyester agar plates are very
efficient methods in the evaluation of the population of polymer-degrading
microorganisms in the environment [35]. By applying the clear zone method, it was
confirmed that the population of aliphatic polyester-degrading microorganisms at 30
and 50°C decreased in the order of PHB = PCL > PBS > PLA [41].
1.4.1. Microorganisms degrading PLA
It reported that 39 bacterial strains of class Firmicutes and Proteobacteria
isolated from soil were capable of degrading aliphatic polyesters such as PHB, PCL,
and PBS, but no PLA-degrading bacteria were found [47]. These results showed
that PLA-degrading microorganisms are not widely distributed in the natural
environment and thus, PLA is less susceptible to microbial attack in the natural
environment than other microbial and synthetic aliphatic polyesters [53].
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In the environment, it is hydrolyzed into low molecular weight oligomers
and then mineralized into CO
2
and H
2
O by the microorganisms presented. Soil
burial tests show that the degradation of PLA in soil is slow and that it takes a long
time for degradation to start. For instance, no degradation was observed on PLA
sheets after 6 weeks in soil [36]. It reported that the molecular weight of PLA films
with different optical purity of the lactate units (100% L and 70% L) decreased by
20 and 75%, respectively, after 20 months in soil [64]. On the other hand, PLA can
be degraded in a composting environment where it is hydrolyzed into smaller
molecules (oligomers, dimers, and monomers) after 45–60 days at 50–60°C. These
smaller molecules are then degraded into CO
2
and H
2
O by microorganisms in the
compost [64].
Recently, several PLA-degrading microorganisms, their enzyme and
substrate specificities have been reported. Microbial degradation of PLA was first
published using an actinomycete Amycolatopsis strain isolated from soil [40]. The
time course of PLLA film degradation by this strain was investigated. The residual
film was recovered from the culture broth by chloroform extraction and about 60%
of the 100 mg film was degraded after 14 days cultivation. However, the strain
could not metabolize further the degradation products as indicated by a low cell
growth and no decrease in the water-soluble total organic carbon (TOC) in the
culture broth [40]. Since then, quite a number of Amycolatopsis strains have been
isolated as PLA degraders. In a report, 50 samples were collected from soil, pond,

and rivers but only two strains were capable of degrading more than 50% of PLLA
film in the liquid medium [15]. The two strains were identical and phylogenetic
analyses showed that the sequence of the strain is closely related to Amycolatopsis
mediterranei with similarities of 96.9% [15]. Another PLLA-degrading
microorganism, Amycolatopsis sp. strain K104-1 was isolated from 300 soil
samples. This strain formed clear zones on the PLLA emulsified agar plates and
was able to degrade more than 90% of 0.1% emulsified PLLA after 8 days [31]. In
addition to Amycolatopsis, several actinomycetes belonging to Lentzea,
Kibdelosporangium, Streptoalloteichus, and Saccharothrix are also capable of
degrading PLLA [10], [19]. These PLLA-degrading actinomycetes are belong to
PLA family Pseudonocardiaceae and related genera [2], [18].
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Screening of PLA-degrading fungi was carried out [62]. Out of 14 fungal
strains tested, only two strains of F. moniliforme and one strain of Penicillium
roqueforti could assimilate lactic acid and racemic oligomer products of PLA but no
degradation was observed on PLA. To date, Tritirachium album is the only PLLA-
degrading fungus that has been reported so far. PLLA degradation increased
significantly upon addition of 0.1% (w/v) gelatin to the culture medium [17].
Microbial degradation of PLLA at high temperature has been reported. A
thermophilic strain, Brevibacillus sp. (formerly Bacillus brevis) which degrades
PLLA film at 60°C was isolated from soil [59], [59]. PLLA-degrading thermophiles
were isolated from a garbage fermentor [43]. One of the isolates, identified as
Bacillus smithii grew well in the medium containing 1% PLLA and the molecular
weight of PLLA decreased by 35.6% after 3 days incubation with shaking at 60°C.
A newly isolated PLLA-degrading thermophile Geobacillus sp. strain 41 was
reported [61]. The time course of PLLA degradation was monitored at 60°C for 20
days and degradation was confirmed by the change in molecular weight and
viscosity of the residual polymer. The PLLA-degrading activity of this strain was

higher than that of Brevibacillus sp. However, it is not clear whether microbial
degradation of PLLA proceeds at elevated temperatures ≥55°C (higher than Tg of
PLLA) because under this condition, PLLA can be easily hydrolyzed at a relatively
high rate [39].
The assimilation of PLLA degradation products by microorganisms is also
an important mechanism because it is an indication that these water-soluble
substances could be mineralized in natural and artificial environments as long as
suitable microbial populations are present [62]. In addition, the degradation
products could be used by the microorganisms for their growth and eventually
metabolized to CO
2
and H
2
O. The degradation and assimilation must occur at a
sufficiently rapid rate so as to avoid accumulation of materials in the environment.
Most of the PLLA-degrading strains are able to assimilate the degradation products
[53], [62].
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1.4.2. Microorganisms degrading PHB
The ability to degrade PHB is widely distributed among bacteria and fungi:
Aerobic and anaerobic PHB-degrading microorganisms have been isolated from
various ecosystems such as soil, compost, aerobic and anaerobic sewage sludge,
fresh and marine water (including deep sea), estuarine sediment, and air [20], [21].
It reported for the first time the PHB-degrading microorganisms from Bacillus,
Pseudomonas and Streptomyces species [8]. From then on, several aerobic and
anaerobic PHB-degrading microorganisms have been isolated from soil
(Pseudomonas lemoigne, Comamonas sp. Acidovorax faecalis, Aspergillus
fumigates and Variovorax paradoxus), activated and anerobic sludge (Alcaligenes

faecalis, Pseudomonas, Illyobacter delafieldi), seawater and lake-water
(Comamonas testosterone, Pseudomonas stutzeri) [26], [55].
The percentage of PHB-degrading microorganisms in the environment was
estimated to be 0.1-10% of the total colonies [35]. Majority of the PHB-degrading
microorganisms were isolated at ambient or mesophilic temperatures and very few
of them were capable of degrading PHB at higher temperature. It emphasized that
composting at high temperature is one of the most promising technologies for
recycling biodegradable plastics and thermophilic microorganisms that could
degrade polymers play an important role in the composting process [55]. Thus,
microorganisms that are capable of degrading various kinds of polyesters at high
temperatures are of interest. A thermophilic Streptomyces sp. Isolated from soil can
degrade not only PHB but also PES, PBS and poly[oligo(tetramethylene succinate)-
co-(tetramethylene carbonate)] (PBS/C). This actinomycete has higher PHB-
degrading activity than thermotolerant and thermophilic Streptomyces strains from
culture collections [6]. A thermotolerant Aspergillus sp. was able to degrade 90% of
PHB film after five days cultivation at 50°C [44]. Furthermore, several thermophilic
polyester degrading actinomycetes were isolated from different ecosystems. Out of
341 strains, 31 isolates were PHB, PCL and PES degraders and these isolates were
identified as members of the genus Actinomadura, Microbispora, Streptomyces,
Thermoactinomyces and Saccharomonospora [55], [63].