screening and study on microorganisms degrading biopolymers in vietnam

Study degradation of biodegradable polymers by isolated strains .... The topic: "Screening and study on microorganisms degrading biopolymers in Vietnam" was therfore inplemented in order

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

2.4 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

3.3.1.1 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

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

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

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

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

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

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

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 CO2 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

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

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]

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]

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

1.2.1.1 Poly(Lactic Acid) (PLA)

O O

O O

O n

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

1.2.1.2 Poly(3-Hydroxybutyrate) (PHB)

PHB ([-O(CH3)CHCH2CO-]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]

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

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]

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]

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]

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

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

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]

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 CO2 and H2O [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]

In the environment, it is hydrolyzed into low molecular weight oligomers and then mineralized into CO2 and H2O 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 CO2 and H2O 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]

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 CO2 and H2O 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]

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 co-(tetramethylene carbonate)] (PBS/C) This actinomycete has higher PHB-

succinate)-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]

1.4.3 Microorganisms degrading PCL

PCL has been shown to be degraded by the action of aerobic and anaerobic microorganisms that are widely distributed in various ecosystems Furthermore, the

degradation of high molecular weight PCL was investigated using Penicillium sp

strain 26-1 (ATCC 36507) isolated from soil PCL was almost completely degraded

in 12 days This strain can also assimilate unsaturated aliphatic and alicyclic polyesters but not aromatic polyesters [52] A thermo-tolerant PCL-degrading

microorganism which was identified as Aspergillus sp strain ST-01 was isolated

from soil PCL was completely degraded by this strain after 6 days incubation at 50°C [44] PCL and PHB were degraded under anaerobic condition by new species

of microorganisms belonging to the genius Clostridium [1] PCL can be degraded

by lipases and esterases [57] The degradation rate of PCL is dependent on its molecular weight and degree of crystallinity Enzymatic degradation of PCL by

Aspergillus flavus and Penicillium funiculosum showed that faster degradation was

observed in the amorphous region [9], [55]

The biodegradability of PCL can be increased by copolymerization with aliphatic polyesters In general, copolymers have lower crystallinity and lower Tm than homo-polymers, and are thus more susceptible to degradation The susceptibility of PCL films which were prepared at different quenching

temperatures (-78, 0, 25, 50°C) by R arrhizus lipase was evaluated at 30°C Large

spherulites were formed on PCL film quenched at 50°C but no spherulites were observed on PCL film quenched at -78 °C X-ray diffraction diagram of PCL films quenched at 25°C and 50°C indicated the growth of PCL crystal in the direction of c-axis (thickness of crystal unit), but crystallinity did not increase so much (about 40%) with the rise in quenching temperature The susceptibility of PCL film quenched at -78°C was the highest and the susceptibility decreased with increase in quenching temperature It was confirmed that the size of spherulites is an important factor for biodegradation of PCL In addition, the storage modulus of PCL film samples increased with increase in quenching temperature PCL film quenched at 50°C had the highest storage modulus, while low storage modulus was observed on PCL film quenched at -195°C Storage modulus of PCL film also increased with

increase in draw ratio [55] Furthermore, the susceptibility of PCL films to R

arrhizus lipase decreased with increase in draw ratio As the storage modulus of

polyesters can be determined over a wide range (from below Tg up to Tm), we could predict the rate of enzymatic degradation of polyesters by using the value of storage modulus of polyesters at 30°C (the typical evaluation temperature of biodegradability) [54]

Chapter 2: MATERIALS AND METHODS

2.1 MATERIALS

24 soil samples were collected from different locations in Hanoi: Mai Dich – Cau Giay, My Dinh-Tu Liem, Cau Dien – Tu Liem, Tay Mo – Tu Liem Samples varied depending on the environment, such as farm soil, compost, soil at riverside… Samples were collected under the surface about 10 - 20 cm, at the area rich of plastic waste

PLA powder was gifted from Research Center for Environmental Technology and Sustainable Development, Hanoi University of Science was used in this study with the number-average molecular weight (Mw) of 3.5104 or PLA granules were purchased from Polysciences, Inc., USA with the number-average molecular weight of 5104 composed of 100% L-lactic acid PHB powder, was obtained from Aldrich Co., USA with the number-average molecular weight was 2105 PCL granules were obtained from Wako Co., Japan, with the number-average molecular weight 4104 Biopolymer film was obtained from Jassco Inc., Japan

2.2 CHEMICALS

Agar mineral salt medium (MSM) medium overlaid by PLA, PHB or PCL were used for isolation polymers-degrading microorganisms Basal mineral salt medium contained (g/l): MgSO4, 0.1; NaCl, 0.1; KH2PO4, 0.2; CaCl2, 0.02; FeSO4, 0.01; yeast extract, 0.1; and supplemented 17 g agar The medium was sterilized at

121oC for 20 minutes It was cooled to 60oC and poured into plates To overlay polymers film onto agar plates, polymers were added to chloroform solution (0.01 g/ml) and 250 µl solution was spread on the agar plates [22]

Agar MSM medium containing emulsified polymers were used for screening polymers-degrading microorganisms Polymers emulsion was prepared by dissolving 2g PLA, PHB or PCL in 20 ml chloroform by sonication (50W, 15 min)

in 1 liter MSM medium [50] Agar 2% (w/v) was added to the emulsified medium

and dissolved by heating which stimulated the evaporation of chloroform The agar medium was autoclaved at 121oC for 20 minutes and then poured into petri dishes

LB medium was used for culture bacteria composed of (g/l): Peptone, 10; Yeast extract, 5; NaCl, 10; agar, 17-20

Gause medium was used for culture actinomycetes composed of: (g/l)

K2HPO4, 0.5; MgSO4, 0.5; KNO3, 1; NaCl, 0.5; FeSO4, 0.01; agar, 17-20; Starch,

20 The pH was adjusted to 7.2-7.4

Czapek medium was used for culture fungi containing (g/l): NaNO3, 2;

KH2HPO4, 1; KCl, 0.5; MgSO4, 0.5; FeSO4, 0.01; NaCl, 0.5; agar, 20; glucose, 30 The final pH was 7

Other chemicals for study: Tris-base, ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), phenol, chloroform, isopropanol, ethanol, sodium acetate… were high purification for research purposes

2.3 EQUIPMENTS

Equipments were used for study composed of: Shaker (Satorius-Germany), Spectrophotometer (Bionate-English), PH meter (Horiba, Japan), Kern Scale (Satorius-Germany), Warm cabinet (Memmert, Germany), Sigma 3K30 centrifugor (Sartorius, Germany), Desiccator (Memmert, Germany), Scanning electron microscope JSM-5421LV (Japan), TOC-VCSH analyzer (Shimadzu, Japan)…

All the equipments were used at Key Laboratory of Enzyme-Protein Technology, Institute of Microbiology and Biotechnology, 69 Institute- President

Ho Chi Minh Mausoleum Protection High Command, Faculty of Biology, Hanoi University of Science, Vietnam National University

2.4 METHODS

2.4.1 Isolation of biopolymer-degrading microorganisms

In order to isolate strains have polymers degradable activity, firstly soil samples were activated to enrich the number of colonies in the sample 5 mg each samples was added to 50 ml liquid MSM in 100-ml flasks supplemented with 0.1% PLA, PHB or PCL (w/v) and cultured on a rotary shaker 160 rpm at 37oC After 1

week, 0.1 were volumes transferred to fresh medium and incubated for another 2-3 days

The enrichment culture broth was diluted with (0.9%) NaCl solution and spread on agar MSM plates that were overlaid with PLA, PHB or PCL film and incubated at 37oC Polymers were overlaid onto plates as described Biopolymers-degrading strains grew on the plates after 2-5 days were collected and were purified

by repeated transfers to LB agar plates [42]

2.4.2 Screening biopolymer-degrading microorganisms

A very simple semi-quantitative method was the clear-zone test This was an agar plate test in which the polymer is dispersed as very fine particles within the synthetic medium agar; this results in the agar having an opaque appearance Agar MSM medium containing emulsified polymers were used for this method Strains were incubated on biopolymer-emulsified agar plates for 3 days After inoculated with microorganisms, the formation of a clear halo around the colony indicated that the organisms were at least able to depolymerize the polymer [35]

Because clear zones formed on PHB or PCL were not clearly, another method to screening polymer degrading microorganisms is measurement the growth

of these strains in the medium supplemented polymers as sole sources of carbon Strains were grown on 5ml test tube MSM medium supplemented PHB/PCL (0.1%) The development of the isolated strain showed the degrading ability of this strain with the polymer On the other hand, the development of the isolated strain was determined by count plate method

2.4.3 Identification of biopolymers-degrading strains

2.4.3.1 Gram staining method

Principle of this method based on differences in the biochemical composition

of bacterial cell walls Gram-positive bacterial walls are rich in tightly linked peptidoglycans (protein-sugar complexes) that enable cells to resist de-colorization Gram-negative bacterial walls have a high concentration of lipids (fats) that dissolve

in the de-colorizer (alcohol, acetone, or a mixture of these) and are washed away

with the crystal violet The de-colorizer thus prepares gram-negative organisms for the counterstain [12]

Procedure

- Make a smear of each strain on the slide

- Flood slide with crystal violet Allow to stand for one minute

- Flood with Gram’s iodine (a mordant) Leave for one minute

- Wash off with tap water

- Decolorize with alcohol (95%) until no more color washes off (usually 10–

20 seconds) This is a most critical step Be careful not to over decolorize, as many gram-positive organisms may lose the violet stain easily and thus appear to be gram negative after they are counterstained

- Wash off with tap water

- Apply safranin (the counterstain) for one minute Then wash off with tap water

- Drain and blot gently with bibulous paper Air-dry the slide thoroughly before you examine the preparation under the microscope

2.4.3.2 Observation under scanning electron microscopy (SEM)

Observation strains at large magnifications could determine clearly morphology and dimension of cell It also distributed to classify species so SEM observation method was applied The principle of this method was the samples were coated with gold using a JOEL, JFC-1200 fine coater before observation

Procedure

Bacteria were inoculated in the plates for 24 hours before observing

- Pick up single colony, add to Glutaraldehyte solution in 2 hours

- Centrifuge at 3000 rpm for 10 minutes at 4oC

- Rinse with CaCodylate buffer 2 times

- Fixed cells by 1% Osmic acid solution (OsO4) for 1 hour

- Centrifuge at 13,000 rpm for 10 minutes at 4oC

- Rinse again by CaCodylate buffer

- Remove water by ethanol with different concentrations: 30o, 50o, 70o, 90o,

100o Each concentration in 10 minutes

- Create a well on the sole and drop the sample above into the well

- Sample was golden-plated on JFC-1200 machine in 30 seconds at 30 mA

- Scanning on the electron microscopy JSM-5421LV (Japan) in 69 Institute- President Ho Chi Minh Mausoleum Protection High Command

2.4.3.3 Extraction of genomic DNA from bacteria

Genomic DNA extraction was carried out using procedures described

Procedure of extraction DNA

- The bacteria were cultured in LB environment for 24 hours

- Centrifuge at 6,000 rpm for 10 minutes to collect cells

- Add 40 µl Proteinase K and shake horizontally at 37oC for 30 min

- Add 600 µl SDS (20%), incubate at 65oC for 2h with gentle end over end

inversion every 25 minutes

- Centrifuge at 5000 rpm for 10 minutes at 20oC

- Collect supernatant and add 400 µl phenol: chloroform: isoamyl alcohol 25:24:1 ratio Mix thoroughly and centrifuge at 13000 rpm for 10 minutes Collect aqueous phase and precipitate DNA by addition of 0.6 volume of isopropanol Incubate at room temperature for 1h then centrifuge at 13.000 rpm for 20 min at 20oC

- Decant supernatant and dry pellet under vacuum (or in air)

- Resuspend DNA in 50 μl TE, store at -20oC Check the quality and concentration of the extracted DNA on agarose gel 1% (in TAE)

2.4.3.4 Amplification of the 16S rDNA

The 16S rDNA gene was amplified by PCR using forward and reverse primers which annealed at positions 8-27 and 1543-1525 respectively

27f: (5’-AGAGTTTGATCCTGGCTCAG-3’)

1525r: (5’-AAAGGAGGTGATCCAGCC-3’)

PCR procedure

- Obtain a 0.2 ml PCR tube which contains 40 µl of the PCR master mix Label this tube with your initials

- Add 10 µl of your DNA sample to the PCR master mix (total volume: 50 µl)

- The tube can then be place in the PCR machine

The negative control was used for each PCR experiment is that DNA template was altered by water

The PCR machine has been programmed as follows:

The PCR product was purified by ethanol precipitation

2.4.3.5 Agarose gel electrophoresis

The DNA genome and PCR products will be analysed by agarose gel

electriophoresis A 1.5% agarose gel will be prepared The loading buffer will

contain ethidium bromide This dye will intercalate between the DNA strands this complex will be flourescent under UV light This allows the DNA bands to be visualized and photographed Molecular weight markers of known sizes (bp) are included in the agarose gel electrophoresis run In this study, marker 1.5 kb was used

2.4.3.6 Sequencing

16S rDNA sequencing reactions were read by ABI PRISM 310 Sequencer (U.S.A) at the Institute of Microbiology and Biotechnology The 16S rDNA sequences were initially analyzed by using program BLAST (National Center for Biotechnology Information) Result of analyzed sequence was compared with other

species in the international gene bank by Clustal X software Phylogenetic tree is built based on Treeview software.

2.4.3.7 Effect of culture conditions (temperature, pH, NaCl concentration)

Study effect of temperature, pH and NaCl concentration were not only helped to optimize growth conditions of researched strain but also supported to categorize into taxonomy groups, so we carried to determine the effect of these factors on the growth of researched strain

To optimize growth NaCl concentration, the strainswere cultured for 1 day in flask with 20 ml of MSM medium supplemented glucose instead of polymers The media containing various concentrations of NaCl: 0%, 0.1%, 0.5%, 1%, 5% to evaluate the effect of NaCl concentrations on the growth of isolated strains [23]

Test to optimize growth pH was determined at pH 4, 5, 6, 7, 8, 9 in a similar manner pH was controlled by adding HCl 0.1% or NaOH 0.1% using pH meter

To observe the effects of temperature on the growth of strains, cultures were incubated at different temperatures: 4oC, 25oC, 37oC, 50oC, 65oC [42]

Cell growth in broth above was measured by the absorbance 660 nm using spectrophotometer

2.4.3.8 Utilization of sugars

This is a characteristic that also supports to classify researched strains Strain was cultured in a test tube using 5 ml of MSM medium containing kinds of sugar: D (+) Galactose, D (+) Glucosamin, L (-) Fucose, D (+) Mannose, Lactose, L (+) Arabinose, Mannosamine, N-acetyl D-Glucosamin, D (-) Fructose, D-Glucose, L-Rhamose with the concentration was 1% (w/v) at 37oC with shaking at a rate of 120 rpm The optical density of the broth was then measured at 660 nm using spectrophotometer after 2 days

2.4.3.9 Activity of some extracellular enzymes

Amylase and cellulase activities were investigated based on the hydrolysis of starch, cellulose on agar MSM plates containing substrates (starch or cellulose 0.1%) [12] Enzyme broths were added to plates and were incubated at 37o

Catalase was assayed by adding diluted H2O2 (3%) onto freshly grown colonies Catalase-positive isolates produce bubbles within a few seconds [12], [42].

Protease activity was briefly described as follows: an enzyme sample (0.3 ml) was added to 1.5 ml of 6 g casein per liter in 50 mM sodium phosphate buffer (pH 7) The reaction mixture was incubated at 30oC for 1h and the reaction was terminated by adding 1.5 ml of 0.11 M trichloroacetic acid solution Filtration was performed before determining the activity at 275 nm One unit of protease activity was defined as the amount of enzyme required to liberate 1µmol equivalent of L-tyrosine per minute [50]

2.4.4 Study degradation of biodegradable polymers by the isolated strains

2.4.4.1 Growth experiment in PLA, PHB or PCL containing media

Degradation of polymer was carried out in 100 ml-flask containing 40 ml liquid MSM medium supplemented 40 mg PLA, PHB or PCL The seed cultures were grown for 1 day in MSM medium before added to experiment flasks (5% volume) The control experiment was carried out which cells were not added Flasks were incubated at 37oC with shaking at 160 rpm [19] The time course of assimilation was monitored by the growth of microorganism as well as the dissolved total organic carbon (TOC) concentration, the polymer weight at different points of time The growth of strain was measured by absorbance at 660 nm using spectrophotometer or by plate count method

2.4.4.2 Measurement of the PLA, PHB or PCL residual weight

Residual polymers were recovered from the culture broth by extraction with

100 ml of chloroform The lower layer, which consisted of chloroform and residual polymer, was collected The combined solvent extracts were dried at 80oC overnight The weight of residual polymers was measured

2.4.4.3 Determination of TOC in culture broth

Dissolved total organic carbon (TOC) in the broth was determined by

TOC-VCSH analyzer (Shimadzu, Japan)

Principle: To determine the quality of organically bound carbon, the organic

molecules must be broken down to single carbon units and converted to single

molecular form that can be measured quantitatively TOC methods utilize heat and oxygen, ultraviolet irradiation, chemical oxidants, or combinations of these oxidants

to convert organic carbon to carbon dioxide (CO2) The CO2 may be measured directly by a non-dispersive infrared analyzer, it may be reduced to methane and measured with a flame ionization detector, or CO2 may be titrated chemically

Procedure:

Set up standard curve:

- Preparation of standard solutions: Accurately weigh 2.125 g of reagent grade potassium hydrogen phthalate and transfer to 1 liter distilled water The carbon concentration of the solution corresponds

to 1000 mg/l (1000 ppm) This solution is diluted with water to prepare other concentrations: 200 ppm, 500 ppm

- Run TOC analyzer with the standard solutions: 0 ppm, 200 ppm, 500 ppm, 1000 ppm

- From the results exported about peak area, we built a standard curve

as follow (Fig 6)

Fig 6 The standard curve of TOC

Measurement dissolved TOC in the broth: Before analysis, the volume of the culture broth was made up to 100 ml with distilled water Five ml sample was taken from the culture broth to measure the dissolved TOC concentration The broth was filtrated through a 0.2 µm membrane and measured upon the standard curve [19]

2.4.4.4 Degradation experiment with biopolymer film

Degradation of polymer was carried out in 100 ml-flask containing 40 ml liquid MSM medium with biopolymer film added The seed cultures were grown for

1 day in MSM medium before added to experiment flasks (5% volume) The control experiment was carried out which cells were not added Flasks were incubated with shaking at 160 rpm The result was determined by the growth of microorganisms in the medium

2.4.4.5 Statistical analysis

Data from experiments were compiled as files in Excel All experiments were repeated at least 3 times to check reproducibility

Chapter 3: RESULTS AND DISCUSSION

3.1 ISOLATION AND SCREENING PLA, PHB, PCL-DEGRADING MICROORGANISMS

Twenty-four soil samples, which were collected from many sites in Hanoi, were used in this study These samples were activated for 1 week and then screened

on agar MSM medium overlaid with PLA, PHB and PCL for isolation PLA, PHB and PCL-degrading microorganisms After 3-5 days, colonies were formed on the plates The clear zone was also formed around each colony containing organism capable of degrading the polymer The clear zones on plates varied in size and clarity (Fig 7) The size and clarity of the clear zone apparently reflected ability of microorganisms to degrading biopolymers Based on the clear zones appeared and the differences in colony morphology, strains were collected On PLA agar plates,

11 bacterial strains, 3 mold strains were collected 21 strains on PHB medium and

18 strains on PCL medium were also collected

Fig 7 Colonies isolated on PLA- overlaid medium

On the selection medium, microbial species which exhibit the growth under the chosen conditions, but these organisms are not necessarily the most efficient polymer-degrading strains [50] Thus, these strains were screened once again in the polymer emulsified media On PLA emulsified plates, of 14 strains only 5 strains could form clarity degrading-zones (Fig 8) Among these strains, strains G5 and Cz1 formed the largest clear-zones, so these two strains were chosen to further studies

Fig 8 Strains growth on PLA emulsified plate

On the other hand, of 21 strains there were 8 strains formed the clear zones

on PHB emulsified agar plates and of 18 strains only 6 strains could form clear zones on PCL emulsified agar plates; the rest of them was not Clear zones performed were small and not clarity, so it was difficult to compare the degrading ability of these strains (Fig 9)

Fig 9 Strains growth on (a) PHB and (b) PCL emulsified plate

With the aim to have specific strains to study further, PHB and degrading strains were grown in test tubes containing 5 ml MSM medium supplemented PHB and PCL (0.1%), respectively After 3 days inoculation, the growth of these strains were determined by plate count method and the result showed that strain B2 grew best in the PHB medium and strain B1 was in PCL medium, respectively (Table 3.1) Therefore, these strains also were studied further

C5