SCREENING AND CHARACTERIZATION OF PHAPRODUCING BACTERIA FROM ACTIVATED SLUDGE

HIMI HUSME BIN ABD MAJID

UNIVERSITI TEKNOLOGI MALAYSIA

PSZ 19:16 (PIND 1/07)

UNIVERSITI TEKNOLOGI MALAYSIA

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Authors full name : HIMI HUSME BIN ABD MAJID Date of birth Title : : 29 APRIL 1986 SCREENING AND CHARACTERIZATION OF PHA-PRODUCING BACTERIA FROM ACTIVATED SLUDGE

Academic Session:

2007/2008-2

I declare that this thesis is classified as :

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(Contains confidential information under the Official Secret Act 1972)* (Contains restricted information as specified by the organization where research was done)* I agree that my thesis to be published as online open access (full text)

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If the thesis is CONFIDENTAL or RESTRICTED, please attach with the letter from the organization with period and reasons for confidentiality or restriction.

SCREENING AND CHARACTERIZATION OF PHA-PRODUCING BACTERIA FROM ACTIVATED SLUDGE

HIMI HUSME BIN ABD MAJID

A report submitted in partial fulfillment of the requirements for the award of the degree of Bachelor of Science (Pure Biology)

Faculty of Science University Technology Malaysia

APRIL 2007

ii

I hereby declared that I have read this thesis and in my opinion this thesis is sufficient in terms of scope and quality for the award of the degree of Bachelor of Science (Pure Biology).

Signature

: ... : 16th April 2008

Name of Supervisor : Dr. Adibah Yahya Date

iii

I hereby declare that this thesis entitled Screening and Characterization of PHAProducing Bacteria from Activated Sludge is the result of my own research except as in cited references. The thesis has not been accepted for any degree and is not concurrently submitted in candidature of any other degree

Signature Name Date

: .. : Himi Husme Bin Abd Majid : 16th April 2008

iv

In loving memory of my dad, Abd. Majid Bin Mohd. Salleh For my beloved and supporting mom, Normah Mamat, my dearest sister, Nadiratul Noziana and my step dad, Azmi bin Abdul Wahab. .Thank you very much

ACKNOWLEDGEMENT

Firstly, I would to give my gratitude to my project supervisor, Dr. Adibah Yahya, for her commitment, time spend, encouragement, support and guidance in completion of this project throughout the year. It would be difficult for me to complete this project without her help and advice. Next, I would also like to thank Assc. Prof. Dr. Zaharah Ibrahim for her advice and ideas and also the caring attitude all throughout the year while completing this project. A lot of thank to lab assistants, Puan Fatimah, Puan Radiah, Encik Awang and Encik Yus for their cooperation in providing all the lab instruments and guidance while using the instruments. Not to forget Cik Fareh Nunizawati that has given me her advice and support to do the experiments efficiently. I would also like to express my appreciation and thanks to my loving and caring parents, my lovely sister and all the people and friends that help me complete this project. Thank you very much

vi

ABSTRACT

Eleven strains of bacteria previously isolated from activated sludge in PHA-producing reactor were used in this study. The activated sludge contains mix culture of bacteria that were able to use various types of organic compound presence in the sludge for growth and PHA (polyhydroxyalkanoate) production. In this study, the bacteria were screened for their ability to produce PHA in a minimal medium supplemented with glucose as carbon source and grow at 30oC. Results indicated that all bacteria tested were able to produce PHA, though only four strains showed enhanced PHA production. However, only two strains coded strain 2 and strain 5 were selected for further identification due to higher and almost similar concentration of PHA produced which is 1.4909 mg/L and 1.4935 mg/L respectively. Morphology characteristic of strain 2 and strain 5 showed that both were clearly distinguish by their shape of colonies. Strain 2 was observed and showed white flat colony with clear edge zone at each of the colony. In contrast, the colony of strain 5 appeared to be more like fungi and was highly slimy. Cellular characterization showed that strain 2 was a rod shape cell and strain 5 was coccobacilli cell. From the biochemical test, strain 2 may belonged to Bacillus species and strain 5 can be a Acinetobacter species. The 16s rRNA characterization of the bacteria showed that strain 2 also belonged to Bacillus species (82%).

vii

ABSTRAK

Sebelas jenis bakteria yang sebelumnya dipencilkan daripada selut beraktivasi di dalam reaktor yang menghasilkan PHA telah digunakan dalam kajian ini. Selut beraktivasi tersebut mengandungi campuran kultur bacteria yang berkebolehan untuk menggunakan pelbagai jenis bahan organik yang terdapat di dalam selut untuk pertumbuhan dan penghasilan PHA (Polyhydroxyalkanoate). Dalam kajian ini, bakteria tersebut dikesan kebolehannya untuk menghasilkan PHA dalam medium minimum yang ditambah dengan glukosa sebagai sumber karbon dan tumbuh pada 30oC. Keputusan menunjukkan semua bakteria yang diuji berkebolehan untuk menghasilkan PHA. Walaupun begitu, hanya empat jenis bakteria mununjukkan penghasilan PHA yang tinggi. Walaubagaimanapun, hanya dua jenis bakteria berkod strain 2 dan strain 5 telah dipilih untuk pengenalpastian lanjutan merujuk kepada penghasilan PHA yang lebih tinggi dan kepekatan yang hampir sama iaitu 1.4909 mg/L dan 1.4935 mg/L setiapnya. Pencirian morfologi bagi strain 2 dan strain 5 menunjukkan kedua-duanya dapat dibezakan degan jelas berdasarkan bentuk koloni mereka, Strain 2 telah diperhatikan dan menunjukkan koloni putih rata dengan zon lutsinar pada setiap koloni. Sebaliknya, koloni bagi strain 5 adalah menyerupai fungi dan sangat berlendir. Pencirian pada peringkat sel menunjukkan strain 2 berbentuk rod dan strain 5 berbentuk rod dan bulat. Daripada keputusan biokimia, strain 2 dikenalpasti tergolong dalam spesis Bacillus dan strain 5 dari spesis Acinetobacter. Daripada pencirian menggunakan teknik 16S rRNA, strain 2 dikenalpasti tergolong dalam sepsis Bacillus (82%).

viii

CONTENTS

CHAPTER

TITLE

PAGE

Title Supervisors Declaration Declaration Dedication Acknowledgements Abstract Abstrak Contents List of Tables List of Figures List of Abbreviation List of Appendices

i ii iii iv v vi vii viii xii xiii xv xvii

INTRODUCTION

LITERATURE REVIEW 2.1 2.2 2.3 2.4 2.5 2.7 2.8 Polyhydroxyalkanoates (PHA) Chemistry of the PHAs Physical Properties of PHAs The biology of PHA Biosynthesis of PHA Recovery of PHA Carbon Substrate and Yield

3 3 4 5 7 7 10 10

ix 2.9 2.10 Other Types of PHA and Application Characterization and Identification 2.10.1 16S rRNA 2.10.2 Polymerase Chain Reaction 2.10.3 Agarose Gel Electrophoresis 2.10.4 Gram Staining 2.10.5 Biochemical Characterization 12 13 13 13 14 15 16

MATERIALS AND METHODS 3.1 3.2 Experimental Design Microorganisms 3.2.1 3.2.2 3.2.3 3.3 Morphology Characterization of Bacteria Preparation of Inoculums Preparation of Stock Culture

18 18 19 19 20 20 20 21 21 21 22

Media preparation 3.3.1 3.3.2 3.3.3 Nutrient Broth Nutrient Agar Mineral Salt Medium

3.4 3.5

Screening of PHA Producer Characterization and Identification of Potential PHA Producing Bacteria 3.5.1 Biochemical Characterization 3.5.1.1 Catalase Test 3.5.1.2 Cytochrome oxidase 3.5.1.3 Nitrate reduction 3.5.1.4 Citrate Test 3.5.1.5 Triple Sugar Iron Test 3.5.1.6 Starch hydrolysis 3.5.1.7 OF-Glucose Test 3.5.1.8 Gelatin liquefaction Test 3.5.1.9 Urease Test

23 23 23 23 24 24 25 25 25 26 26

x 3.5.1.10 Indole Test 3.5.1.11 Motality 3.5.1.13 Lipase Test 3.5.1.14 Voges Proskauer Test 3.5.2 Molecular Characterization 3.5.2.1 DNA Extraction 3.5.2.2 PCR Amplification of 16S rRNA Fragment 3.5.2.3 Purification of the Amplified 16S rRNA Fragment 3.5.2.4 Agarose Gel Electrophoresis 3.5.2.5 Sequencing of the Amplified 16S rRNA Fragment 3.5.2.6 Phylogenetic Tree Construction 3.6 Analytical Methods 3.6.1 3.6.2 Determination of PHA Determination of Bacterial Growth 31 31 32 32 32 30 31 29 26 27 27 27 28 28

RESULT AND DISCUSSION 4.1 4.2 Screening of Potential PHA Producer Colony and Cellular Morphologies Characterization 4.2.1 4.3 4.4 4.5 Gram Staining

33 33 35 40 41 46

Biochemical Characterization Bacterial growth analysis PCR Amplication Method for Microbial Identification 4.5.1 4.5.2 4.5.3 Genomic DNA Extraction PCR Amplication of 16s rRNA Gene Fragment Purification and Qualitative PCR Product analysis 4.5.4 DNA Sequence analysis

47 47 49

50 51

xi 4.5.4.1 Blastn Analysis 4.5.4.2 ClustalX 51 52

CONCLUSION AND FUTURE WORK 5.1 5.2 Conclusion Future Work

54 54 54

REFERENCES

55

APPENDICES

60-71

xii

LIST OF TABLES

TABLE NO.

TITLE

PAGE

2.1

Classification of microbial PHAs according to different criteria

2.2

Effect of substrate cost and P(3HB) yield of the production cost of P(3HB) 11 12 29 30 30

2.3 3.1 3.2 3.3 4.1

Possible application of PHA Sequences of eubacterial 16S rDNA universal primers PCR reaction mixtures Gradient PCR cycle profile Colony morphologies of pure colony on Agar plate culture.

37 40

4.2

Cellular characterization of strain 2 and strain 5

4.3 4.4

Summary of Biochemical test result Quantitative analysis of mixed culture genomic DNA

41 49

xiii

LIST OF FIGURES

FIGURE NO.

TITLE

PAGE

2.1 2.2

General structural formula of PHA Principle of biosynthesis of bacteria in bacteria. Three relevant phases of biosynthesis of PHA are shown

9 18 34 38 38 38 38 38 38 39 39 39 39 39 40 40 42 42 43 43 44

3.1 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 4.13 4.14 4.15 4.16 4.17 4.18 4.19

Schematic representation of overall experimental setup Amount of PHA produce by bacterial strains Colony morphology of Strain 1 Colony morphology of Strain 2 Colony morphology of Strain 3 Colony morphology of Strain 5 Colony morphology of Strain 6 Colony morphology of Strain 8 Colony morphology of Strain 9 Colony morphology of Strain 11 Colony morphology of Strain 12 Colony morphology of Strain 13 Colony morphology of Strain 14 Cellular morphology of strain 2 Cellular morphology of strain 5 Nitrate reduction test of Strain 2 Nitrate reduction test of Strain 5 Starch hydrolysis test on Strain 2 Starch hydrolysis test on Strain 5 Negative result of urease test of strain 2

xiv 4.20 4.21 4.22 4.23 4.24 4.25 4.26 Positive result of urease test of strain 5 Gel liquefaction test of strain 2 Gel liquefaction test of strain 5 No growth on MacConkey agar of strain 2 Growth on MacConkey agar of strain 5 Cell dry weight analysis plot of strain 2 and strain 5 Agarose gel analysis of genomic DNA (1% w/v agarose, 80 volts, 45 watts, 60 minutes) 4.27 Agarose gel analysis of PCR products (1% w/v agarose, 80 volts, 45 watts, 60 minutes) 4.28 Agarose gel electrophoresis of the purified PCR products (1% w/v agarose, 80 volts, 45 watts, 60 minutes) 4.29 Phylogenetic tree processed and illustrated by Tree View 51 52 50 48 44 45 45 46 46 47

xv

LIST OF ABBREVIATIONS

A BLAST bp
o

Absorbance Basic Local Alignment Search Tool base pair degree Celcius distilled water double stranded DNA Deoxyribonucleic acid deoxynucleotide triphosphate Ethylenediaminetetraacetic acid Extracellular polysaccharide Sulphuric acid Hydrogen chloride gram per litre kilobase pairs microlitre minutes hours mililitre Molar nanogram per microlitre nanometer National Center of Biotechnology Information sodium hydroxide Hydroxyl percent

dH2O dsDNA DNA dNTP EDTA EPS H2SO4 HCl g/L kbp L min h mL M ng/L nm NCBI NaOH OH %

xvi PCR PHA rRNA sp. Ta Tm UV V w/v v/v Polymerase Chain Reaction Polyhydroxyalkanoate ribosomal Ribonucleic Acid species Annealing temperature Melting temperature Ultra violet Volts weight per volume volume per volume

xvii

LIST OF APPENDICES

APPENDIX

TITLE

PAGE

OD at 600nm analysis, cell dry weight and ln X value of strain 2 and strain 5 60 62 62 62

B B B C

OD 600nm analysis plot of strain 2 and strain 5 ln X analysis plot of strain 2 ln X analysis plot of strain 5 Value of OD and amount of PHA produce by bacterial strains

63 64 65 66 67 68 68 69 70 71

D E F G H H I J K

Beef extract peptone broth Preparation of OF-Glucose Medium Preparation of Triple Sugar Iron (TSI) Agar Preparation of Simmons Citrate Agar Preparation of Lugols Iodine Preparation of Kovacs reagent Preparation of Christensen urea agar slant Preparation of Tryptone Broth medium Preparation for lipase activity

CHAPTER 1

INTRODUCTION

In response to increasing public concern about the harmful effects of petrochemical derived plastic materials in the environment, many countries are conducting various solid-waste management programs, including plastic waste reduction by developing biodegradable plastic materials. These biodegradable plastic materials must retain the desired material properties of conventional synthetic plastics and should be completely degraded without leaving any undesirable residues when discarded.

Polyhydroxyalkanoates, (PHAs) are polyesters of various hydroxyalkanoates which are synthesized by numerous microorganisms as an energy reserve material, usually when an essential nutrient such as nitrogen or phosphorus is limited in the presence of excess carbon source. PHAs are considered to be strong candidates for biodegradable polymer material because they possess material properties similar to various synthetic thermoplastics and elastomers currently in use (from polypropylene to synthetic rubber) and upon disposal, they are completely degraded to water and carbon dioxide (and methane under anaerobic conditions) by microorganisms in various environments such as soil, sea and lake water.

This study was purpose for finding the best bacteria that grow and produce high concentration of polyhydroxyalkanoate, (PHA). PHA are same polymer as plastics but it can it degrade more and effectively than normal plastics that are made from petroleum. Although many bacteria can produce PHA when supplied with the suitable growth condition and carbon substrate, not all the bacteria can produce high production of PHA.

2 The objectives of this study are: 1. 2. To screen the potential PHA-producing bacteria. To characterize selected potential bacteria using biochemical and molecular method

CHAPTER 2

LITERATURE REVIEW

2.1

Polyhydroxyalkanoates (PHA)

Polyhydroxyalkanoates (PHAs) are the polymers of hydroxyalkanoates that accumulate as carbon/energy or reducing-power storage material in various microorganisms (Salehizadeh and Van Loosdrecht, 2004). PHAs are stored in the bacterial cytoplasm as inclusion bodies (Lee, 1996) and they are synthesized and accumulated intracellularly as distinct granules, usually under unfavorable growth conditions, such as feast and famine regime, limitation of nitrogen, phosphorus, sulphur, magnesium or oxygen in the presence of excess carbon source (Poirier, 1995).

Basically, PHAs can be broadly subdivided into three groups based on the number of carbon atoms present in its monomer units (Steinbuchel, 2001),:

(a) Short-chain-length PHAs consisting of 3-5 carbon atoms (PHA

SCL

).
MCL

(b) Medium-chain-length PHAs consisting of 6-14 carbon atoms (PHA (c) Long-chain-length PHAs consisting of more than 14 carbon atoms (PHA
LCL

).

).

4 2.2 Chemistry of the PHAs

Of all the biodegradable plastics being studied, those that have generated the most interest are the poly(3-hydroxyalkanoates) or PHAs which are made by bacteria. Like all plastics, PHAs are polymers, long molecules made up of many small subunits (monomers) which have been joined together. These water-insoluble storage polymers are biodegradable, exhibit thermoplastic properties and can be produced from renewable carbon sources. The composition of the polymer synthesized is governed by two main factors, i.e. the bacterial strain being used and the carbon source utilized to grow the bacteria. (S.P. Valappil, 2007)

In the case of PHAs, the monomers are 3-hydroxyalkanoates. An alkanoate is simply a fatty acid which is a linear molecule containing just carbon and hydrogen (an alkane) with a carboxyl group at one end (making an alkanoate). Furthermore, these monomers have a hydroxyl group (OH) at the 3rd carbon (what used to be called the beta position), making these beta or 3-hydroxyalkanoates. The hydroxyl group of one monomer is attached to the carboxyl group of another by an ester bond; these plastics are thus polyesters.

As is shown in Figure 2.1, the polyester linkage creates a molecule which has 3carbon segments separated by oxygen atoms. The remainder of the monomer becomes a sidechain off the main backbone of the polymer. Most of the PHAs encountered in nature are poly(beta-hydroxybutyrate) (PHB), in which the monomer unit is hydroxybutyric acid and the side chain is a methyl group. Other monomer units occur in nature, and many others can be produced in the laboratory by feeding unusual carbon sources to bacteria. Most PHAs, even what we call PHB, are actually copolymers, and contain some amount of another type of monomer unit.

Figure 2.1

General structural formula of polyhydroxyalkanoate (PHA)

2.3

Physical Properties of PHAs

The composition of the PHA has a direct effect on the physical properties of the plastic, in. PHB, with its short methyl side chain, is a very crystalline and very brittle polymer. Industrially, it is difficult to use because the temperature at which it melts is very close to the temperature at which it begins to decompose. Its high degree of crystallinity causes it to crack easily. As a result, the PHA used commercially is PHBV, a copolymer of hydroxybutyrate and hydroxyvalerate (5 carbons long). PHBV is a random copolymer, meaning that the monomer units do not occur in the chain in any particular order. PHBV can still crystallize, but it produces a much more supple plastic and melts at a lower temperature, making processing easier. PHB and PHBV have properties similar to polypropylene, and bottles made from these polyesters feel just like "normal" plastic. The flexibility increases with sidechain length throughout the PHA family, largely because of a loss of crystallinity. Polymers composed mostly of hydroxyoctanoate, an 8- carbon monomer, are elastic. Longer side chain polymers are so soft that they are gummy or glue-like. This remains one of the potential values of PHAs, that by feeding bacteria an appropriate substance, a PHA with specific desirable properties can be produced.

6 PHAs can be classified into various groups according to different criteria (Table 2.1). Among them, classification according to the monomer size, which refers to the number of carbon atoms in the HA monomer, and the type of polymer is the most common and will be described in detail here.

Table 2.1 : Classification of microbial PHAs according to different criteria (Luengo, 2003) Natural PHAs: produced naturally by microorganisms from general substrates. i.e Poly(3-hydroxybutyrate) P(3HB). Biosynthetic origin Semisynthetic PHAs: requires the addition of unusual precursors such as 3-mercaptopropionic acid to promote the biosynthesis of poly(3-hydroxybutyrateco-3-mercaptopropionic) [P(3HB-co-3MP)]. Monomer size (depending on the number of carbon atoms in an HA monomer Short chain-length PHAs (SCL-PHA): contains 3-5 carbon atoms. Medium chain-length PHAs (MCL-PHA): contains 614 carbon atoms. Homopolymer: The polymerization begins with the linkage of a small molecule or monomer through ester bonds to thecarboxylic group of the next Number of different monomers in PHAs monomer. A homopolymer is produced when single monomeric units are linked together. i.e P(3HB). Heteropolymer: When two or more different

monomeric units are linked together, a copolymer is formed. i.e P(3HB-co-4HB). Chemical nature of the monomers PHAs containing aliphatic fatty acids. i.e P(3HB). PHAs containing aromatic fatty acids. PHAs containing both aliphatic and aromatic fatty acids. i.e P(3HB-co-3MP).

7 2.4 The Biology of PHA

In nature, prokaryotic microorganisms respond to sudden increases in essential nutrients in their usually hostile environment by storing important nutrients for survival during prolonged period of starvation (Sudesh, 2000). PHAs are one such storage compound. PHAs are usually produced when carbon sources are in excess. The carbon sources are assimilated, converted into hydroxyalkanoate (HA) compounds and finally polymerized into high molecular weight PHAs and stored as water insoluble granules in the cell cytoplasm. PHAs are an excellent storage compound because their presence in the cytoplasm, even in large quantities does not disturb the osmotic pressure of the cell.

PHA granules can be observed as refractile granules under phase contrast light microscope. When thin sections of cells containing PHAs are viewed under transmission electron microscope, the granules appear as electron transparent, discrete, spherical particles with clear boundaries. The number and sizes of granules per cell differ depending on the PHA-producer microorganisms and their growth stage. In Wautersia eutropha (formerly known as Alcaligenes eutrophus), 8-13 granules per cell with sizes ranging from 0.2-0.5 m were detected (Byrom, 1994).

PHA granules could be stained with Sudan Black (Schlegel, 1970) and more specifically by Nile Blue A, exhibiting a strong orange fluorescence. Nile Blue A is a more specific dye than Sudan Black B as it does not stain glycogen and polyphosphate. Both stains however can stain lipid bodies.

2.5

Biosynthesis of PHA

Polyhydroxyalkanoates are polyesters of hydroxyalkanoates (HAs) having the general structural formula shown in Figure 2.1. Numerous bacteria can synthesize and accumulate PHAs as carbon and energy storage materials or as a sink for redundant

8 reducing power under the condition of limiting nutrients in the presence of excess carbon (Steinbuchel, 1991).

Three metabolic phases of the biosynthesis of PHA in bacteria can be distinguished (Figure 1.3). First, a carbon source suitable for biosynthesis of PHA must enter the cell from the environment. This is achieved either by a specific transport system located in the cytoplasmic membrane or by diffusion of the compound into the cell. Second, anabolic or catabolic reactions, convert the compound into a hydroxyacyl coenzyme A thioester which is a substrate of the PHA synthase. Third, PHA synthase, which is the key enzyme of PHA biosynthesis, uses these thioesters as substrates and catalyzes the formation of the ester bond with the concomitant release of coenzyme A. At present it cannot generally be excluded that the PHA synthases also use other thioesters of HA as substrates. Phase II is of most importance, since during this phase the carbon source is converted into a suitable substrate for the PHA synthase. Many bacteria are able to convert acetyl-CoA in two steps via acetoacetyl-CoA to D(-)-3hydroxybutyryl-CoA giving rise to poly(3HB). Regarding the application of precursor substrates, the most simple type of reaction is the conversion of a HA, which is provided as a carbon source to the cells, by a thiokinase or a coenzyme A transferase into the corresponding HA-coenzyme A thioester, such as, for example, the conversion of 4HB into 4-hydroxybutyryl-coenzyme A. If the carbon source is not a precursor substrate, and if the carbon source is first converted into a central intermediate of metabolism, a complex sequence of reactions may be required to obtain PHA consisting of HA other than 3HB (Steinbiichel. 1995).

9 Figure 2.2: Principle of biosynthesis of bacteria in bacteria. Three relevant phases of biosynthesis of PHA are shown

2.6

Production of PHA

Figure 2.2

Principle of biosynthesis of bacteria in bacteria. Three relevant phases of

biosynthesis of PHA are shown. (Steinbiichel. 1995).

In most bacteria PHAs are synthesized and intracellularly accumulated under unfavorable growth conditions such as limitation of nitrogen, phosphorus, magnesium, or oxygen in the presence of excess carbon (Anderson, 1990). It is, therefore, important to develop cultivation strategies that can simulate these conditions for the efficient production of PHA.

Some bacteria such as Alcaligenes latus and a mutant strain of Azotobacter vinelandii are known to accumulate PHA during growth in the absence of nutrient limitation. Selection of a microorganism for the industrial production of PHA should be based on several factors including the cells ability to utilize an inexpensive carbon source, growth rate, polymer synthesis rate, and the maximum extent of polymer accumulation. The yield of PHA on carbon source is important not to waste substrate to non- PHA material. An equation that predicts the overall yield of PHA on several carbon sources has been derived and can be used for the preliminary calculation of PHA yields (Yamane, 1992). Recovery of PHA should also be considered because it significantly affects the overall economics.

10 2.7 Recovery of PHA

Following the fermentation, cells containing PHAs are separated by conventional procedures such as centrifugation, filtration, or vortexing. After the biomass harvesting, cells are disrupted to recover the polymers. A number of different methods have been developed for the recovery of PHA. The first method that has most often been used involves extraction of P(3HB) from biomass with solvent. The solvents employed include chloroform, methylene chloride, propylene carbonate, and dichloroethane. Due to the high viscosity of even dilute PHA solutions, about 20 portion of solvent are required to extract 1 portion of polymer (Byrom, 1994). The large amount of solvent required makes this method economically unattractive, even after the recycling of the solvent (Holmes, 1994).

Several other methods that have been developed involve the use of sodium hypochlorite for the differential digestion of non-PHA cellular materials (Berger, 1989). Although this method is effective in the digestion of non-PHA cellular materials, it causes severe degradation of P(3HB) resulting in a 50% reduction in the molecular weight. The use of sodium hypochlorite together with chloroform significantly reduced degradation of PHA (Hahn, 1994). It was suggested that chloroform immediately dissolves the isolated P(3HB) by hypochlorite, and thus protects polymer from degradation. Normally, polymer purity of greater than 95% is obtained by hypochlorite treatment.

2.8

Carbon substrate and yield

Excluding the recovery process, the economics of PHA production are largely determined by the substrate cost and PHA yield. The efficiency of substrate conversion is important, and can be predicted from the physiology and biochemistry involved in the PHA synthesis. Among the various nutrients in the fermentation medium, the carbon source contributes most significantly to the overall substrate cost in PHA production. A

11 number of carbon sources, including carbohydrates, oils, alcohols, acids and hydrocarbons, can be used by various bacteria (Yamane, 1993).

The theoretical yield (the yield based on the reaction stoichiometry) of P(3HB) has been estimated for several of these carbon substrates (Yamane, 1993). Regeneration of nicotinamide nucleotides, which are used as cofactor for PHA synthesis, has been taken into account in this analysis. It was also suggested that the overall yield, which is the yield in actual fermentation, would be roughly proportional to the theoretical yield and PHA content.

Table 2.2 summarizes the cost of carbon substrate based on the theoretical yield. Because of their low price, crude carbon substrates such as the cane and beet molasses, cheese whey, plant oils and hydrolysates of starch (corn and tapioca), cellulose and hemicellulose can be excellent substrates to several bacteria utilizing them.

Table 2.2 : Effect of substrate cost and P(3HB) yield of the production cost of P(3HB) Approximate price (US$/kg) 0.493 0.290 0.180 0.595 0.502 0.220 0.071

Substrate Glucose Sucrose Methanol Acetic acid Ethanol Cane Molasses Cheese Whey

P(3HB) yield 0.38 0.40 0.43 0.38 0.50 0.42 0.33

Substrate cost 1.30 0.72 0.42 1.56 1.00 0.52 0.22

12 2.9 Other types of PHA and application

Beside PHA, there are many types of PHA. The material properties and hence the application of the PHAs vary depending on the monomers composition. Polyhydroxybutyrate, P(3HB), is the most well known and well characterized PHA. However, industrial applications of P(3HB) have been hampered knowing to its low thermal stability and excessive brittleness upon storage (Lee, 1996). The copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV), P(3HB-co-3HV), is more flexible and tougher than the P(3HB). It can be used to make various products, including films, coated paper, board, compost bags, disposable food service ware and moulded products such as bottles and razors and also be used for biomedical applications (Lee, 1996).

Besides that, there have recently discovered 4-hydroxybutyrate, P(4HB). The P(4HB), has been found to be useful in the biomedical applications (Martin and Williams, 2003). It was used for tissue engineered heart valve scaffold and viable ovine blood vessels (Chen and Wu, 2005). Also, a high molecular weight copolymer of 3HB and 4HB [P(3HB-co-4HB)] containing 0100 mol% of 4HB, can be produced by Comamonas acidovorans with a controlled degradation rate (Saito and Doi, 1994), making them ideal candidates for biomedical applications such as tissue engineering (Martin and Williams, 2003). Other application of PHA are shown in Table 2.3.

Table 2.3: The possible application of PHA Packaging films, bags and containers Biodegradable carrier for long term dosage of drugs, medicines, insecticides, herbicides, or fertilizers Disposable items such as razors, utensils, diapers, or feminine hygiene products Surgical pins, sutures, staples, and swabs Wound dressing

13 2.10 Characterization and identification of bacteria

2.10.1 16S rRNA

Prokaryotic classification has historically relied on phenotypic attributes such as size and shape, staining characteristics, and metabolic capabilities to group organisms. Newer molecular techniques such as DNA sequencing give a greater insight into the evolutionary relatedness of microorganisms. DNA sequences are viewed as evolutionary chronometers, meaning that sequence differences appear to provide a relative measure of the time elapsed since the organisms diverged from common ancestor (Willey, 2008).

DNA sequencing enables one to more accurately construct a phylogenetic tree. These trees are somewhat like a family tree, tracing the evolutionary heritage of organisms. Each line or branch of the tree represents the evolutionary distance between two species. Individual species are represented as nodes.

2.10.2 Polymerase Chain Reaction

The first step is to synthesize DNA fragment with sequence identical to those flanking the targeted sequence. This is accomplished with a DNA synthesizer. These synthetic oligonucleotides are usually about 20 nucleotides long and serve as DNA primer DNA synthesis. The primers are one component of the reaction mixture, which also contains the target DNA, a thermostable DNA polymerase, and each of the four deoxyribonucleoside triphosphates (dNTPs). PCR requires a series of repeated reactions, called cycles. Each cycle has three step that are precisely executed in a machine called thermocycler (Willey, 2008).

In the first step, the target DNA containing the sequence to be amplified is heat denatured to make it single stranded. Next, the temperature is lowered so that the

14 primers can hydrogen bond or anneals to the DNA on the both sides of the target sequence. Because the primers are very small and are present in excess, the targeted DNA strands anneal to the primer rather than to each other. Finally, DNA polymerase extends the primers and synthesizes copies of the target DNA sequence using dNTPs. Only polymerase able to function at high temperatures employed in the PCR technique can be used.

At the end of one cycle, the targeted sequences on both strands have been copied. When the three step cycle is repeated, the two strands from the first cycle are copied to produce four fragments. These are amplified in the third cycle to yield eight doublestranded products. Thus each cycle increases the number of target DNA molecules exponentially. After approximately 30 cycles of PCR, the DNA region flanked by the primers will have been amplified approximately a billion-fold (Nester, 2007)

2.10.3 Agarose Gel Electrophoresis

Agarose Gel Electrophoresis is a method used in biochemistry and molecular biology to separate DNA, RNA or protein molecules by size. In gel electrophoresis, charged molecules are placed in an electric field and allowed to migrate toward the positive and negative poles. The molecules separate because they move at different rates due to their differences in charge and size. Because DNA is negatively charged , it is loaded into wells at the negative pole of the gel and migrates toward the positive. Each fragments migration rate is inversely proportional to the log of its molecular weight. That is to say, the smaller a fragment is, the faster it moves through the gel. Migration rate is also a function of gel density. In practice, this means that higher concentration of gel material provide better resolution of small fragments and vice versa (Willey, 2008)..

DNA that has not been digested with restriction enzymes is usually supercoil. For this other reasons, DNA is usually cut with restriction endonucleases prior to

15 electrophoresis. Small DNA molecules usually yield only a few bands because there are few restriction enzyme recognition sites. If the DNA fragment is large, or an entire chromosome is digested, many such sites are present and the DNA is cut in numerous places. When such DNA is electrophoresed, it produces a smear representing many thousands of DNA fragments of similar sizes that cannot be individually resolved.

The DNA is not visible in the gel unless it is stained. To do this, the gel containing the separated DNA fragment is immersed in a solution containing ethedium bromide. This dye binds DNA and fluoresces when viewed with UV light. Each fluorescent band represents millions of molecules of specific-sized fragment of DNA (Nester, 2007).

2.10.4 Gram Staining

The gram stain is a useful stain for identifying and classifying bacteria. The Gram stain allows you to classify bacteria as either gram positive or gram negative. The Gram staining technique was discovered by Hans Christian Gram in 1884 when he attempted to stain cells and found that some lost their color when access stain was washed off.

The staining technique consists of applying primary stain which is crystal violet, applying Grams iodine, applying ethyl alcohol which acts as decolorizing agent and applying secondary stain or counter stain which is safranin.

The most important determining factor in the procedure is that bacteria differ in their rate of decolorization. Those that decolorize easily are referred to as gram negative, whereas those that decolorize slowly and retain the primary stain are called gram positive.

16 The Gram stain is most consistent when done on young cultures of bacteria (less than 24 hours old). When bacteria die, their cell walls degrade and may not retain the primary stain, giving inaccurate results. Because Gram staining is usually the first step in identifying bacteria, the procedure should be memorized.

2.10.5 Biochemical Characterization

Enzymatic activities are widely used to differentiate bacteria. Even closely related bacteria can usually be separated into distinct species by subjecting them to biochemical test, such as one to determine their ability to ferment an assortment of selected carbohydrates. For one example of the use of biochemical tests is to identify bacteria. Moreover, biochemical tests can provide insight into a species niche in the ecosystem. For example, a bacterium that can fix nitrogen gas or oxidize elemental sulfur will provide important nutrients for plants and animals (Nester, 2007).

CHAPTER 3

MATERIALS AND METHODS

3.1

Experimental design

The main aim of this study is to screen and identify the potential polyhydroxyalkanoate (PHA) producer from the bacterial strains (Section 3.2) previously isolated from an activated sludge bioreactor for PHA production. Several experimental activities were scheduled in order to ensure a successful achievement of the project aim (Figure 3.1). The bacterial strains were revived from glycerol stock cultures by growing at 30oC in nutrient broth medium (Section 3.3.1). The culture purity was checked by streaking the culture onto nutrient agar medium (Section 3.3.2). Screening of the potential PHA producer was carried out by growing the pure bacterial culture into a defined medium (Section 3.3.3) commonly used for PHA production. The PHA production was monitored using spectrophotometer technique (Section 3.6.1). Selected bacteria that show the highest PHA production were further used for bacterial identification using biochemical (Section 3.5.1) and biomolecular (Section 3.5.2) methods.

18 14 strains of bacteria are grown on Nutrient Agar for 24 h After bacteria growth

Each strain are transferred to each conical flask that contained mineral salt media and cultivated for 5 days. .

Each bacteria were checked for the production of PHA using the spectrophotometer

Bacteria that produced the highest production of PHA were selected for further analysis

(Phylogenetic tree contruction)

(Biochemical test)

DNA extraction

14 test were done

PCR amplification

Purified PCR Product

Sequencing

Figure 3.1 : Schematic representation of overall experimental setup

19 3.2 Microorganisms

Eleven strains of bacteria coded strain 1, strain 2, strain 3, strain 5, strain 6, strain 8, strain 9, strain 11, strain 12, strain 13 and strain 14 were obtained from the culture collection of Research Laboratory 2, Department of Biology, Faculty of Science, Universiti Teknologi Malaysia, Skudai, Johor. The bacteria stored as glycerol stock cultures at -20oC were revived by inoculating into universal bottles containing 5 mL of sterile nutrient broth medium (Section 3.3.1) and incubated at 30oC for 24 h without shaking. The purity of each bacterial culture was monitored by streaking onto separate nutrient agar medium (Section 3.3.2) and incubated for 24 h at the same temperature. The bacterial colonies grown on the solid medium were then observed under stereo microscope (Leica model CME microscope). Culture observed with colonies of the same morphology on nutrient agar is considered pure culture, whereas those observed with two or more types of colonies are subjected to culture purification via single colony isolation method. A single colony of different morphologies were aseptically selected, streaked onto a separated Nutrient agar medium and allowed to grow at 30oC for 24 h. Repeated single colony isolation was carried out until the culture purity was ensured.

3.2.1

Morphology characterization of bacteria

Pure cultures of bacteria were streaked onto Nutrient agar medium (Section 3.3.2) and incubated at 30oC for 24 h. The bacterial colonies formed on the surface of the medium were then observed using a stereo microscope to record for their morphology such as shape and pigmentation.

20 3.2.2 Preparation of inoculums

The pure bacterial strains were streaked onto the Nutrient agar medium (Section 3.3.2), incubated at 30oC for 24 h and pure colony was aseptically transferred into sterile universal bottles containing 15 mL of the Nutrient broth medium (Section 3.3.1). The bacteria were allowed to grow in a shaking incubator at 30oC, 200rpm for 24 h. The cultures turbidity was measured using a bench top spectrophotometer (Jenway 6300) at the wavelength of 600nm. Cultures with the absorbance values ranging from 0.7-0.8 were used as inoculums.

3.2.3

Preparation of Stock Culture

Pure culture of bacteria were inoculated (10% v/v) into universal bottles containing 10 mL of sterile Nutrient broth medium (Section 3.3.1) at 30oC for 24 h. A 0.8 mL of the fresh cultures were then transferred into a separate sterile Eppendorf tube and added with 0.2 mL glycerol to a final volume of 15% v/v. The cultures were then frozen in liquid nitrogen prior to place at -80oC freezer for long term storage.

3.3

Media preparation

Solid and liquid media used in this study are of enriched and defined types. All media are prepared as described in section 3.3.1 to 3.3.3.

21 3.3.1 Nutrient Broth

This is an enrichment medium used to grow the bacteria when preparing the inoculums. Nutrient broth has been a commonly used medium to grow heterotrophic bacteria. In this study, the medium was used for the preparation of bacterial inoculums. Nutrient broth consists of peptone, meat extract and distilled water. An 8 g/L of Nutrient broth powder was added into a 1L Schott bottle containing 1L of distilled water. They were mixed and sterilized via autoclaving at 121oC for 20 min. The broth was left to cool to 50oC prior to pour into sterile universal bottle. The universal bottle can be directly used or stored at 4oC for subsequent use.

3.3.2

Nutrient Agar

Nutrient agar is commercially obtained which consisted of peptone and beef extract as the source of carbon, minerals and vitamins which are important to support bacterial growth; sodium chloride as the carbon source of chloride ion and agar as gelling agent. A 20.0 g/L of the nutrient powder was added into a 1L Schott bottle containing 1L of distilled water. They were mixed and sterilized via autoclaving at 121oC for 20 min. The medium were left to cool at 50oC prior to pour into sterile Petri plates. The agar was left to solidify at room temperature. The plates can be directly used or stored at 4oC for later used. All plates were sealed with parafilm in order to avoid contamination during storage.

3.3.3

Mineral Salt Medium

For PHA production from all the bacterial strain, the strain was grown in mineral salt media containing 10 g/L glucose and microelement. The mineral salt media consisted of (g/L) 0.5 (NH4)2SO4, 0.4 MgSO4.7H2O, 9.65 Na2HPO4.12H2O, 2.65

22 KH2PO4 in distilled water. All the content was autoclaved at 121oC for 20 min except for MgSO4.7H2O. The MgSO4.7H2O was filter sterilized and added to the autoclave mineral salt media. For the microelement, 1mL from the solution containing 20g FeCl3.6H2O, 10g CaCl2.H2O, 0.03 CuSO4.5H2O, 0.05 MnCl2.4H2O and 0.1 ZnSO4.7H2O in 0.5M HCl was added to the mineral salt media.

3.4

Screening of PHA Producer

This screening method was used for detection of PHA production after cultivation in mineral salt media. The purpose of this is to check or screen which bacterial strain can produce high production of PHA.

Before continuing the screening, centrifuge tubes were first washed with ethanol and hot chloroform to remove and substance or plasticizers. The cultures then were centrifuged at 4000 x g for 30 minutes. The cell pellet or paste was suspended in a volume of commercial sodium hypochlorite solution (Clorox) equal to original volume of medium. After 1 h at 37oC, the lipid granule were centrifuged, wash with water and then wash with alcohol and acetone. The polymer was dissolved with 5 mL of chloroform and left overnight at 28oC on a shaker at 150rpm. Then the contents were centrifuged at 4000 x g for 30 minutes. The supernatant was taken and transferred into clean test tube. The supernatant containing chloroform is evaporated and 5 mL of concentrated H2SO4 were added. The tube is capped and heated for 10 min at 100oC in water bath. The solution is cooled to room temperature and the samples are transferred to silica cuvette and the amount of PHA was determined at 235nm.

23 3.5 3.5.1 Characterization and identification of potential PHA producing bacteria Biochemical Characterization

Enzymatic activities are widely used to differentiate bacteria. Even closely related bacteria can usually be separated into distinct species by subjecting them to biochemical tests, such as one to determine their ability to ferment an assortment of selected carbohydrates. Moreover, biochemical tests can provide insight into a species niche in the ecosystem.

3.5.1.1

Catalase Test

This test is particularly useful in distinguishing staphylococci and micrococci, which are catalase-positive, from streptococci and enterococci, which are catalasenegative The test was done aseptically by picked up bacterial cell from colony of slant growth with an inoculating loop. A drop of hydrogen peroxide was pipette onto the mass of bacterial cells. The drops of hydrogen peroxide were observed to see if bubbles were involved. The production of gaseous bubbles indicates the presence of catalase.

3.5.1.2

Cytochrome oxidase

The oxidase test is a test used in microbiology to determine if a bacterium produces certain cytochrome c oxidases Strains may either be oxidase positive (OX+) or negative (OX-). OX+ normally means that the bacterium contains cytochrome c oxidase and can therefore utilize oxygen for energy production with an electron transfer chain.

A piece of filter paper was moistening in a Petri dish with a few drops of a freshly prepared 1% (w/v) solution of tetramethy-p-phenylenediamine dihydrochloride. A loopful of bacterial growth was aseptically transferred from agar medium and smear it

24 on the moisten paper. The development of violet or purple colour after observing the filter paper within 10 seconds indicates a positive test.

3.5.1.3

Nitrate reduction

A nitrate test is a chemical test used to determine the presence of nitrate ion in solution. The test was perform by inoculate bacterial cultures onto tubes containing beef extract-peptone broth (Appendix D). The tubes were incubated at 37oC for 2-5 days. The tubes were then observed and the displacement of the liquid is indicative of the production of nitrogen. 2-3 drops of Reagent A (Appendix D) and 2-3 drops of Reagent B (Appendix D) were added to each tube. Any immediate red colour demonstrates the presence of nitrite and is indicative of reduction of nitrate and nitrite.

3.5.1.4

Citrate Test

Simmons citrate agar tests the ability of organisms to utilize citrate as a carbon source. Simmons citrate agar contains sodium citrate as the sole source of carbon, ammonium dihydrogen phosphate as the sole source of nitrogen and other nutrients. Organisms which can utilize citrate as their sole carbon source use the enzyme citrase or citrate-permease to transport the citrate into the cell. These organisms also convert the ammonium dihydrogen phosphate to ammonia and ammonium hydroxide, which creates an alkaline environment in the medium.

Using a sterile loop, bacterial culture was streak onto citrate slant agar (Appendix G) and was labelled accordingly to each strain. All the tubes were then incubate at 37oC for 4 days and observed any changes occurred.

25 3.5.1.5 Triple Sugar Iron Test

Triple sugar iron agar (TSI) is a differential medium that contains lactose, sucrose, a small amount of glucose (dextrose), ferrous sulfate, and the pH indicator phenol red. It is used to differentiate enteric based on the ability to reduce sulfur and ferment carbohydrates.

A sterile inoculating loop with bacterial culture was streak onto TSI slant agar (Appendix F). Using the same culture, another bacterial culture was stabbed onto another TSI slant agar. The tubes were labelled carefully and incubate it at 37oC for 24 h.

3.5.1.6 Starch hydrolysis

The test involves the breakdown of starch into maltose. Firstly, all the bacterial strain was inoculated by making a single streak down the middle of the plates. The plates contained sterile nutrient agar supplemented with 0.2% (w/v) soluble starch. All the plates were incubated at 37oC for 1 to 5 days. After incubation, the plates were flooded with Lugols iodine (Appendix H). Any clear are indicates the hydrolysis of starch and unchanged starch will stain dark blue.

3.5.1.7 OF-Glucose Test

OF medium is a nutrient semisolid agar containing high concentration of carbohydrate and low concentration of peptone. The peptone will support growth of bacteria that do not use carbohydrate. The test start with using a sterile needle and the bacterial culture was inoculated each with two tubes of OF-glucose media (Appendix E). 2ml of sterile mineral oil was poured over one of the inoculating tube and replaced the

26 cap. The tube was labelled as anaerobic and the other (not added with mineral oil) as aerobic tube. Both tubes were incubated at 37oC for 1 or 2 days.

3.5.1.8 Gelatin liquefaction Test

Many microorganisms produce gelatinase which catalyzes the hydrolysis of the collagen. Bacterial culture was inoculated in the nutrient broth-gelatin tubes and all the tubes was labelled and incubated at 37oC for 10-14 days. The tubes were placed in a refrigerator for 30-60 minutes after incubation. Lack of gelatin hydrolysis will result in liquid consistency of the medium.

3.5.1.9 Urease Test

Urease is an enzyme that catalyzes the hydrolysis of urea, forming ammonia and carbon dioxide. It is found in large quantities in jack beans, soybeans, and other plant seeds, it also occurs in some animal tissues and intestinal microorganisms.

The surface of the Christensen urea agar slant (Appendix I) was inoculated with bacterial strain and was labelled accordingly. All the tubes were incubated at 37oC for 15 days. After the incubation, the appearance of a red violet colour indicates a positive test while a yellow orange colour indicates negative result.

3.5.1.10 Indole Test

Tryptone broth (Appendix 10) tubes were inoculated with bacterial strain. All the tube then incubated at 37oC for 24 to 48 hours. 10 drops of Kovacs reagent (Appendix

27 H) were carefully layered directly onto the top of the broth culture tube. An immediate formation of a red layer at the top of the broth indicates the presence of indole and a yellow or brown colour is a negative test.

3.5.1.11 Motality

Motality test are used to identify the organism or bacteria that are able to move. In this test, positive test indicates the organism is motile which cause turbidity in the medium.

The media contain beef extract (3.0g), peptone (10.0g), NaCl (5.0g) and Agar (4.0g) in 1L of distilled water. The media was autoclaving at 121oC for 15 minutes. Pure culture was stabbed into the medium with a sterile needle to a depth of 1 inch and incubated at 37oC for 1-2 days

3.5.1.13 Lipase Test

The purpose for Lipase test is to determine whether a bacterium produces a lipase that will hydrolyze a neutral fat to fatty acid and glycerol. The medium used for the test is Spirit Blue agar (Appendix K).

3.5.1.14 Voges Proskauer Test

The principle for this test is to detect the production of acetylmethylcarbinol, a natural product formed from pyruvic acid in the course of glucose fermentation. Positive result show pink colour after added -napthol, and 1 mL of 40% KOH.

28 3.5.2 Molecular Characterization

3.5.2.1 DNA Extraction A 5 ml of overnight culture was added into a centrifuged tube. The tube was centrifuged at 13,000 rpm for 3 minutes. The supernatant were removed and discarded. Then, the pellet was resuspended in 480l of 0.5M EDTA. 120l of lysozyme was added gently mixing by inverting the centrifuge tube and later incubated it at 37oC for 1 hour. After 1 hour, the culture was centrifudge at 13,000 rpm for 3 minutes. The supernatant was removed and 600l of nucleic lysis solution was added and mix gently until the cell resuspended. The suspension then incubated at 80oC for 5 minutes to lyse the cell and let it cool to room temperature. 3l of RNase solution was added to the cell lysate and let it mix by inverting the tube for several time. The mixture was then incubated at 37oC for 30 minutes and let it cool to room temperature. A 200l of protein precipitation solution was added to the RNase-treated cell lysate and vortexing it at high speed. Further incubation was perform for the culture on ice for 5 minutes and centrifuged at 13,000 rpm for 3 minutes. The supernatant was transferred into a fresh centrifuged tube which contains 600l isopropanol (at room temperature) and gently mix it by inverting the tube. The tube was centrifuged at 13,000 rpm for 5 minutes. The supernatant was carefully poured and drain the tube on clean absorbent paper. 600l of 70% (v/v) ethanol was added and gently mix it to wash the DNA pellet and followed by centrifuged the tube at 13,000 for 3 minutes. The supernatant was carefully drain and air dried for 10-15 minutes. 100l of DNA rehydration solution was added to the tube and incubated overnight at 4oC.

The success of genomic DNA isolation was determined by agarose gel electrophoresis analysis.

29 3.5.2.2 PCR Amplification of 16S rDNA Fragment

PCR Amplification of 16S rDNA was carried out using universal primers pA or fd1-07 (forward) and pH or rd1-07 (reverse). The sequences of respective primer are shown in table 3.1. To start the PCR, annealing temperature (Ta) of the primer must be known. Usually, the annealing temperature starts at 5oC below the calculated temperature of primer melting point (Tm).

Ta = T m - 5 o C = 2(A+T) + 4(G+C) - 5oC

Table 3.1 : Sequences of eubacterial 16S rDNA universal primers Primers pA pH Sequence 5- AGA GTT TGA TCC TGG CTC AG -3 5- AAG GAG GTG ATC CAG CCG CA -3

Mixture to run the PCR contains PCR Master Mix (Promega), forward and reverse primer, genomic DNA template and nuclease free water. To prepare, genomic DNA template was added accordingly to the concentration of the genomic DNA, primer, PCR Master Mix and nuclease free water was added to the total of 50 L of all the mixture.

30 Table 3.2 : PCR reaction mixtures Reagents DNA Template Forward Primer Reverse Primer 2X PCR Master Mix Nuclease Free Water Total Volumes (L) Strain 2 1 1 1 25 22 50 Strain 5 3 1 1 25 20 50 < 250 ng 0.1 1.0 M 0.1 1.0 M 1X Final Concentration

Table 3.3 : Gradient PCR cycle profile Steps Initial Denaturation Denaturation Annealing Extension Final Extension Temperature 94oC 94oC 50 55oC 72oC 72 C
o

Duration 4 min 1 min 45 seconds 1 min 7 min

3.5.2.3 Purification of the Amplified 16S rDNA Fragment

Purification of the amplified 16S rDNA was done by using Wizard SV Gel & PCR Clean-up System (Promega) according to the manufacturers instructions. The purpose for doing the purification is to remove enzymes, ethedium bromide, mineral oil, agarose, nucleotides, primers, salts, and other impurities from the DNA samples.

Purication method can be done by using two methods; gel dissolving and direct PCR product solution.

31 3.5.2.4 Agarose Gel Electrophoresis

First step in doing agarose gel electrophoresis is to prepare the agarose gel. 1g of agarose was weight out and added into 50ml 1XTAE buffer. The suspension was heated in a microwave until dissolved. The solution was left to cool at about 40oC and ethidium bromide was added into the molten agarose solution. Then, the molten agarose was added into gel molding tray and carefully inserted a comb into the molten gel to allow the formation of well for DNA loading. Carefully, the combs were removed by pulling them upwards firmly and smoothly in a continuous motion.

After finished preparing the gel, the next step is to prepare the DNA sample for electrophoresis. Firstly, a 5l of the DNA sample was aliquot into a microcentrifuged tube. 1l of loading dye was added and mix it by flicking the tube several times. The mixture was loaded into the well. A 10kb mass ruler DNA marker was used as standard for determining the size of DNA fragment. The electrophoresis chamber was closed with lid and connects all power cable according to the colour code. The gel was run at 80V for approximately 1 hour and observed.

3.5.2.5 Sequencing of the Amplified 16S rDNA Fragment

The PCR product that have been purified, were sent to 1st BASE Laboratory Sdn. Bhd., Selangor. Both forward and reverse primer was also included as sequencing probe.

3.5.2.6 Phylogenetic Tree Construction Sequence result obtained from 1st BASE Laboratory Sdn. Bhd., Selangor, were analyzed using online software BLASTn (Basic Local Alignment Search Tool of nucleotide). The sequence match obtain from the BLASTn are subjected to ClustalX

32 program to align the multiple sequence. It calculates the best match for selected sequence and lines them up so that the identities, similarities and differences can be seen. Evolutionary relationships can be seen via tree view application.

3.6 3.6.1

Analytical Methods Determination of PHA

PHA that was extracted from cell was determined by using UVSpectrophotometer. Before the determination, the solution (Section 3.4) was diluted with sterile distiller water. The solution was diluted to the dilution factor of 10. This is because, the initial solution contain concentrated sulphuric acid and cannot be read by the UV-Spectrophotometer.

The absorbance of the UV-Spectrophotometer was set to 235nm and all the absorbance reading of each samples was recorded and check.

3.6.2

Determination of Bacterial Growth

The normal bacterial growth curve has four phases; the lag phase, the log phase, the stationary phase and death phase. This curve is affected by environmental and nutritional factors.

45 mL of nutrient broth with 5 mL of bacterial inoculums was inoculated in conical flask at put in shaker incubator at 200rpm and the temperature was set at 30oC. The time interval to measure the optical densities is every 30 minutes. 1 mL from each flask was transferred to cleaned cuvettes to check the optical densities by using spectrophotometer at 600nm.

CHAPTER 4

RESULTS AND DISCUSSIONS

4.1

Screening of potential PHA producer

Some bacteria have been identified that have the ability to produce PHA. The productions of PHA are depended on the environmental factor, growth condition and the nutrient available. Referring to Wennan He, (1998), PHA production can be enhanced by using mineral salt media. The medium used to grow the bacterial strains can affect the production of PHA from bacteria. By using mineral salt media, the PHA production was successfully produced from the bacteria. The mineral salt media are supplemented with glucose as the carbon source. PHA was produced by the bacteria intracellulaly. During the cultivation of the bacteria, some of the bacterial strain produced slimy and foam. Different kinds of smell were also produced. The PHA produced in the cell were harvested by using dispersion of chloroform and aqueous sodium hypochloride. Before the harvesting of PHA, all the centrifuged tubes were washed with ethanol and hot chloroform. This is to ensure the removal of any plastic material that maybe presence in the centrifuged tubes than can be interfering with the results of screening for PHA production from the cell. During the extraction of the PHA from the cultivated bacterial strains, there are some precaution procedures that must be taken seriously. Because the extraction used solution that are dangerous which are some volatile and corrosive, the use of lab coat, latex glove and mouth mask must be used. Some solution such as hot chloroform, ethanol, and concentrated sulfuric acid must be handling with care.

34 The addition of sulphuric acid in the last step of extraction of the PHA is very concentrated and it cannot be read by UV-Spectrophotometer at 235nm. So, the concentrated sulfuric acid that contains PHA must be diluted first. Silica cuvette was used because the normal cuvette can react with the sulfuric acid and can interfere with the reading of UV-Spectrophotometer.

Figure 4.1

Amount of PHA produce by bacterial strains

35 The amount of PHA extracted was determined as the following (Slepecky and Law, 1960). A = kbc OD = 1.55 x 10-4 x 1 x c x DF c = OD x DF 1.55 x 104 x 1

A = optical density (OD) k b c = molar extinction coefficient of crotonic acid = diameter of cuvette = concentration of PHA

DF = dilution factor

By using the formula, results showed in Figure 4.1 indicated that strains 1, 2, 5 and 6 were among the potential PHA producers. However, two strains which are strain 2 and strain 5 showed highest production of PHA compared to other strains. This could possibly due to the ability of these strain to metabolized glucose more efficiently and resulted to the production of high biomass for the accumulation of PHA. Strain 8, strain 12 and strain 13 showed low production of PHA. This maybe due to the limited amount of cell or they may favor other environmental factor and other carbon source besides glucose. Based on the report by Wennan He, (1998), glucose was also found to be the favorable substrate (carbon source) for PHA production.

4.2

Colony and Cellular Morphologies Characterization

A total of 11 strain of pure colony of bacteria was successfully grow from the culture collection of Research Laboratory 2, Department of Biology, Faculty of Science, Universiti Teknologi Malaysia, Skudai, Johor. All the strains of pure colony show different characteristic in morphologies and sizes.

36 Some of the strains grow very turbid and produce slime which is called EPS. EPS is an extracellular polysaccharide which enables a bacterium to survive by attaching to various surfaces in its natural environment in order to survive. Through attachment, bacteria can grow on diverse surfaces such as rocks, plant roots and even on bacteria. These attachments are due to the present of glycocalyx. Besides giving attachment, the glycocalyx can also protect a cell against dehydration. For example, strain 8 (Figure 4.7) and strain 13 (Figure 4.11). Strain 2 and strain 3 show similarity in the shape of colony. However, strain 2 grow faster and have a clear zone at the end of the colony and differ to strain 3. Many of the strains show same pigmentation colour which is white (strain 1, strain 2, strain 3, strain 5, strain 6, strain 8 and strain 13), yellow (strain 9, strain 11 and strain 12) and pink (strain 14). Most of the strains appeared to have rough surface due to the characteristic of the strains and colony growth. However, for strain 8 and strain 13, the surface of the colony appeared to be smooth due its slimy growth. This could lead to inability for the strains to produce single colony.

37 Table 4.1 : Colony morphologies of pure colony on Agar plate culture.

Colony surface Optical Characterization

Strain

Forms

Elevation

Margins

Pigmentation

filamentous

umbonate

filamentous

rough

white

opaque

circular

raised

undulate

rough

white

translucent

circular

raised

undulate

rough

white

opaque

circular

convex

entire

rough

milky white

opaque

circular

effuse

entire

rough

white

translucent

irregular

umbonate

entire

smooth

milky white

opaque

circular

flat

entire

glistening

yellow

translucent

11

circular

convex

entire

glistening

yellow

opaque

12

circular

convex

entire

rough

light yellow

translucent

13

irregular

umbonate

entire

smooth

white

translucent

14

circular

convex

entire

smooth

pink

translucent

38

Figure 4.2 Colony morphology Strain 1

Figure 4.3 Colony morphology Strain 2

Figure 4.4 Colony morphology Strain 3

Figure 4.5 Colony morphology Strain 5

Figure 4.6 Colony morphology Strain 6

Figure 4.7 Colony morphology Strain 8

39

Figure 4.8 Colony morphology Strain 9

Figure 4.9 Colony morphology Strain 11

Figure 4.10 Colony morphology Strain 12 Figure 4.11 Colony morphology Strain 13

Figure 4.12 Colony morphology Strain 14

40 4.2.1 Gram Staining

Gram staining was done to further characterize the pure colony of the bacterial strains to observe their cellular morphology and arrangement. Strain 2 and strain 5 was selected to be characterize because its ability to produce high amount of PHA. The cellular characteristics of the strains are shown in Table 4.2.

Table 4.2: Cellular characterization of strain 2 and strain 5

Strain 2 Figure 4.27 5 Figure 4.28 Gram Reaction Positive Negative Cellular Arrangement Rod shape Possible related Microbes Bacillus species

Branching coccobacilli (oval Under phylum Acinetobacter and cocci)

Under the microscope, the gram staining results revealed that strain 2 appeared to be gram positive. Its cellular arrangement is rod shape. It can be possibly related to Bacillus species. Meanwhile, strain 5 appeared to be gram negative. Its cellular arrangement is similar to branching coccobacilli which is oval and cocci in shape. It can be possibly related under phylum Acinetobacter.

Figure 4.13 Cellular morphology of strain 2 Figure 4.14 Cellular morphology of strain 5

41 4.3 Biochemical Characterization

Biochemical test are used to determine and to differentiated the bacteria based on their properties. The biochemical tests were catalase, cytochrome oxidase, nitrate reduction, citrate, TSI, starch hydrolysis, OF-Glucose, gelatin liquefaction, urease, indole, lipase, gram staining, MacConkey agar, Voges-proskauer and motility test. The results are summarized in the Table 4.3.

Table 4.3 : Summary of Biochemical test result Biochemical Test Catalase Urease Citrate Nitrate Cytochrome Oxidase Lipase Gel Liquefaction Indole Starch Voges Motality Mcconkey Gram Staining TSI OF-Glucose Strain 2 + + + Fermentation of glucose and lactose Fermentative organism Strain 5 + + + + + + Fermentation of glucose and lactose Fermentative organism

Table 4.3 summaries the biochemical test that have been done on strain 2 and strain 5. From the catalase test, strain 2 show no reaction which give negative result and

42 strain 5 show reaction with production of bubbles which give positive result. Catalase is an enzyme that catalyzes the decomposition of hydrogen peroxide (H2O2) to water and gaseous oxygen. Most aerobic microorganisms possess catalase. The function of catalase is to remove toxic hydrogen peroxide that forms during the oxidation-reductions reactions that are coupled with oxygen in respiratory metabolism. The presence of catalase is shown when hydrogen peroxide is added to a colony or loopful of bacteria and bubbles of oxygen are released from the surface (Jean F, 1980).

For the nitrate reduction test, both of strain 2 and strain 5 showed positive result (Figure 4.15, Figure 4.16). The strain can use nitrate as the terminal electron acceptor in place of oxygen during respiratory metabolism. When this occurs, the pathway is called anaerobic respiration. In anaerobic respiration using nitrate as the terminal electron acceptor, nitratase catalyzes the reduction of nitrate to nitrite. The nitrate may be further reduced to nitrogen gas (Jean F, 1980). The presence of nitrogen gas release from the strains is showed by red color formation.

Figure 4.15 Nitrate reduction test of Strain 2

Figure 4.16 Nitrate reduction test of Strain 5

Strain 2 and strain 5 did not show any positive result to the citrate test. This is because the strains do not utilize citrate as the sole carbon source of carbon. The organisms that utilize citrate and produce an alkaline reaction as indicated by the bromothymol blue, which changes the color from green to blue (Jean F, 1980). Positive result for citrate test is shown by turbidity and blue color changes from green color medium.

43 Triple sugar ion (TSI) is carried out to determine the ability of an organism to attack a specific carbohydrate incorporated in a basal growth medium, with or without the production of gas, along with the possible hydrogen sulfide (H2S) production. Strain 2 and strain 5 show positive results for the fermentation of glucose and lactose (Jean F, 1980).

Starch is a homopolysaccharide, a condensation product of many monomers of a single type of monosaccharide, -D-glucose, forming a polymer of many units united by -glucosidic linkages. Starch hydrolysis test is to check the presence of extracellular amylolitic enzymes that breakdown starch. Strain 2 (Figure 4.17), show negative results for starch hydrolysis test and this showed that it cannot breakdown the starch. Strain 5 (Figure 4.18) showed positive result and was able to breakdown the starch. Positive results on the medium are shown by purple-blue with colorless area around growth of bacteria (Jean F, 1980). Advantage of strain 5 being able to hydrolyze starch are important to producing PHA as it can act to provide the need of other strain that lack this ability.

Figure 4.17 Starch hydrolysis test on strain 2

Figure 4.18 Starch hydrolysis test on strain 5

Oxidative or fermentative organism can be determined by using Rudolph Hugh and Einar Leifsons OF basal media with the desired carbohydrate added. OF medium is a semisolid nutrient agar containing a high concentration of carbohydrate and low concentration of peptone. Fermentation is an aerobic process and bacterial fermenters of a carbohydrate are usually facultative anaerobes. By the fermentation process, a

44 carbohydrate is metabolized and split into two triose carbon molecules (Jean F, 1980). Strain 2 and strain 5 are both fermentative bacteria by the result of OF-Glucose test. Yellow color formation on both medium confirmed the results. Urease test on strain 2 (Figure 4.19), gave negative results howeverpositive for strain 5 (Figure 4.20), wioth red color changes of the medium. The enzyme urease, catalyze the breakdown of urea into ammonia and carbon dioxide. Microorganisms that produce this enzyme are able to detoxify a waste product and derive metabolic energy from its utilization

Figure 4.19 Negative result of urease test of strain 2

Figure 4.20 Positive result of urease test of strain 5

Strain 2 and strain 5 showed positive result of lipase activity. Lipase test are used to determine whether a bacterium produces a lipase that will hydrolyze a neutral fat to fatty acid and glycerol.

Gelatin liquefaction test showed positive result for both strain 2 and strain 5. The strains possess gelatinase which is produced to catalyze the hydrolysis of the protein gelatin (collagen). The hydrolysis of gelatin produces soluble carbohydrates that are readily metabolized as a source of carbon source.

45

Figure 4.21 Gel liquefaction test of strain 2

Figure 4.22 Gel liquefaction test of strain 5

Voges Proskauer test is used to determine the ability of organisms to produce a neutral end product, acetylmethylcarbinol (acetoin), from glucose fermentation. Both strain 2 and strain 5 lacks this ability. Glucose is metabolized to pyruvic acid which is the key intermediate in glycolysis. From pyrivic acid there are many pathways a bacterium may follow. The production of acetoin is one pathway for glucose degradation occurring in bacteria. The ability to hydrolyze tryptophan to indole is a characteristic of strain enteric bacteria that possess the enzyme trytophanase. Trytophanase catalyze the hydrolysis of tryptophan with the production of indole, pyruvic acid and water. However, strain 2 and strain 5 did not have this ability and thus give negative result in the indole test. Strain 2 and strain 5 are nonmotile bacteria from the motality test. The purpose for motality test is to determine if an organism is motile or nonmotile. Bacteria are motile by means of flagella. Flagella occur primarily among the bacilli. However, a few coccal forms are motile. Motile bacteria may contain a single flagellum or many flagella. Nonmotile organisms lack flagella. MacConkey Agar and gram staining is a test that determined and differentiated between gram positive and gram negative bacteria. Growth on MacConkey agar indicates that the bacteria are gram negative bacteria. Strain 5 (Figure 4.24), is gram negative bacteria by means of growth on MacConkey agar and gram staining.

46

Figure 4.23 No growth on MacConkey agar of strain 2

Figure 4.24 Growth on MacConkey agar of strain 5

From the overall biochemical test result, Its showed that strain 2 are related to genus Bacillus species and strain 5 are related under phylum Acinetobacter.

4.4

Bacterial growth analysis

Bacterial growth refers to an increase in bacterial numbers, not an increase in the size of the individual cells. Bacteria normally reproduce by binary fission. Binary fission is a prokaryotic cell reproduction by division into two daughter cells. Once cell division begins, it proceeds exponentially with one cell dividing to form two, each of these cells dividing so that four cell form, and so forth in geometric progression. From Figure 4.25, Strain 2 and strain 5 shows normal growth curve of bacteria. The curves consist of 4 phases which is the lag phase, the log phase, the stationary phase and the death phase. Stationary phase for strain 2 are shorter in time compare to strain 5 but the time for lag and log phase for strain 5 are faster than strain 2. Referring to the cell dry weight analysis, PHA probably being produce at the log phase and as time passed, the availabilty of nutient and biomass accumulated end or less when the phase started to enter stationary phase. The strains started to enter the death phase because nutrient that are needed for the production of PHA by the strains are depleted and the number of deaths exceeds the number of new cells formed.

47

From the ln x analysis against time for strain 2 and strain 5, specific growth rate () for strain 2 is 0.520 (Appendix 2) and specific growth rate for strain 5 is 0.576 (Appendix 2). From the value, its shows that strain 5 can grow faster than strain 2.

Figure 4.25

Cell dry weight analysis plot of strain 2 and strain 5

4.5 4.5.1

16S rRNA Sequencing Genomic DNA extraction

Chromosomal DNA of strain 2 and strain 3 were extracted using Promega WizardTM Genomic DNA Purification Kit. The Kit is an easy and reliable method to purify gram positive bacteria genomic DNA. It is an advanced method compare to the conventional method that use many steps that involve the use of organic solvent. The kit consist of EDTA, lysozyme, Nucleic Lysis Solution, RNase solution, isopropanol, ethanol, DNA Rehydration solution and protein precipitation solution.

48 Lysozyme was used to weaken the cell wall by breaking the -1.4 bonds of peptidoglycan bond. The addition of RNAse was to remove RNA that contaminating the genomic DNA extract. The use of Nucleic Lysis solution is to weaken and lyse the nucleic acid. Protein Precipitation solution was added to remove protein but leaves the high molecular weight genomic DNA in solution. Isopropanol and ethanol would precipitate and concentrate the DNA. DNA Rehydration Solution was added to rehydrate the DNA and was stored at -20oC to prevent from contamination and degradation of the DNA. Agarose gel electrophoresis was used to analyze the isolated genomic DNA to determine the success of the isolation (Figure 4.26). A clear and visible DNA band that showed in lane 1 and 3 indicated that the isolation of genomic DNA of strain 2 and strain 5 were done successfully. The bands that present indicate that the DNA isolated were more than 10,000 bp in size. The visibility of the band obtained shown that the genomic DNA was pure enough for sebsequent PCR amplification purpose. First replicate of strain 2 and first replicate of strain 5 were selected for PCR amplification.

DNA Marker: MassRulerTM DNA Ladder

10000 bp

Mix, 10kbp (Promega) Lane 1: 1st replicate of strain 2 genomic DNA Lane 2: 2nd replicate of strain 2 genomic DNA Lane 3:1st replicate of strain 5 genomic DNA Lane 4: 2nd replicate of strain 5 genomic DNA

Figure 4.26

Agarose gel analysis of genomic DNA (1% w/v agarose, 80 volts, 45

watts, 60 minutes)

49 Purity and concentration of genomic DNA was determined

spectrophotometrically by using a Varian Cary 100 model UV-Vis Spectrophotometer. The concentration of the DNA was determined at the absorbance of 260nm (A260) whereby an optical density (OD) of 1 corresponding to approximately 50 ng/mL of the double stranded DNA. To calculate the purity of genomic DNA, ratio between A260 and A280 (A260/A280) was calculated. The readings of 1.7-2.0 indicate that the genomic DNA is highly purify (Adams, 2003). The ratio (A260/A280) less than 1.7 is considered not pure enough and indicated to the presence of contamination such as protein and phenol. Table 4.4 summarizes the quantitative analysis of genomic DNA.

Table 4.4 : Quantitative analysis of mixed culture genomic DNA Replicate Strain 2 (1) Strain 5 (1) A260 A280 A260/280 1.64 1.20 Dilution factor (DF) 103 103 A260 x DF 26.86 11.67 Concentration (g/l) 1.343 0.584

0.02686 0.01640 0.01167 0.00966

From Table 4.4, result show that the genomic DNA was not pure enough for strain 2 and strain 5 as the ration of A260/A280 was not within 1.7 2.0. This could be cause by contamination that presence in the genomic DNA. The concentration of strain 2 and strain 5 are also low but the PCR amplication were carried out and successfully manage to get the PCR product.

4.5.2

PCR amplication of 16s rRNA gene fragment

Amplifications of the DNA samples were carried out using pA and pH primer. The annealing temperature (Ta) for the primer is 55oC. Based on the genomic isolation, 1 g/L of template DNA from strain 2 and 3 g/L of template DNA was used to start of the PCR reaction. Band on the agarose gel was shown in Figure 4.13 indicated with four

50 clear and strong bands with approximately 1500 bp in length were observed. This indicated that the region of 16S rRNA was successfully amplified using pA and pH primers.

DNA Marker: MassRulerTM DNA Ladder Mix, 10kbp (Promega) Lane 1: 1 replicate of strain 2 Lane 2: 2nd replicate of strain 2
st

1500 bp

Lane 3: 1st replicate of strain 5 Lane 4: 2nd replicate of strain 5

Figure 4.27

Agarose gel analysis of PCR products (1% w/v agarose, 80 volts, 45

watts, 60 minutes)

4.5.3

Purification and qualitative PCR product analysis Purification of the PCR reaction was done using the Wizard SV Gel & PCR

Clean-Up System (Promega). Figure 4.14 show that the success of PCR products from as visualized under UV lamp. The band showed on the agarose gel appeared to be concentrated however replicate one of strain 2 did not show any visible band. This maybe becaused by the lost during the purification step.

51

DNA Marker: MassRulerTM DNA Ladder Mix, 10kbp (Promega) Lane 1: 1st replicate of strain 2 Lane 2: 2nd replicate of strain 2 Lane 3: 1st replicate of strain 5 Lane 4: 2nd replicate of strain 5

1500 bp

Figure 4.28

Agarose gel electrophoresis of the purified PCR products (1% w/v

agarose, 80 volts, 45 watts, 60 minutes)

4.5.4

DNA sequence analysis

The result of purified PCR product for strain 2 and strain 5 after sequencing shows the appearences of multi N-terminal within the sequences. This result occurs because the PCR product that was sent is low in purity and also the concentration.

4.5.4.1

BLASTn analysis

BLASTn performed pairwise comparison of DNA sequences and align to determine the homology of the query sequence. The blasting result for strain 2 showed 100 bases quried. The best scores gives similarity of 82% and this suggested that the bacteria might be closely related to genus Bacillus species. However, strain 5 did not produce any result might due to the sequence result of the purified product.

52 4.5.4.2 ClustalX

After the BLASTn, the sequences were subjected to ClustalX program. ClustalX program combines a good hierarchical method for multiple sequence alignment with an easy to use interface and for preparing phylogenetic tree. Phylogenetic tree is a graphical representation of the evolutionary relationship among a group of organisms or genes. To view the phylogenetic tree produce after running ClustalX analysis of the sequence, Tree View was used. From Figure 4.29, tree showed that strain 2 might be related to Bacillus species that are known can be producing PHA.

Clostridium perfringens ABOO01

Strain 2

Bacillus thuringiensis EF63321

Pseudomonas sp EU557337

Bacillus sp EF633269

Bacillus cereus EF633204

Figure 4.29

Phylogenetic tree processed and illustrated by Tree View

CHAPTER 5

CONCLUSION AND FUTURE WORK

5.1

Conclusion

The efficiency of PHA produce by bacteria depends on the species and how the nutrient is given to the bacteria. Different species of bacteria can be identified from the identification of the shape, color and sizes of the colony produce. Plastics produced from bacteria have become a possible solution to dumping waste because the plastics can breakdown easily as compared to the conventional plastics that were produced from petroleum based compounds.

Using minimal salt media with the supplementation of glucose as carbon source, bacterial strains 2 and 5 produce similar amount of PHA of 1.49 mg/L respectively. Although other strains screened were able to produce PHA, the amount were very low compared to strain 2 and strain 5. The bacterial growth of strain 2 and strain 5 were almost the same but the growth rate of strain 5 appeared to be faster than strain 2.

From the biochemical testing and gram staining results, strain 2 and strain 5 are from Bacillus species and from phylum acinetobacter respectively. Molecular identification supports the result of strain 2. The biochemical tests are able to differentiate the two strains by several tests that have been done.

54 5.2 Future Work

There are several improvements and further study can be suggested: 1. Using several other carbon source and environmental factor such as pH, temperature and availability of oxygen. These factors can be manipulated to check whether the production can be enhanced and high productivity can be accumulated.

2. Genetic engineering method can be introduced in order to higher the limits of production of PHA. The gene responsible for the production can be identified and further studied.

3. Besides researching for production of PHA by bacteria, study can also conducted on the effectiveness of the degradability function of the PHA. PHA can be degraded by nature but time for the degradation can also be account as it can be positive or negative effect on the environment.

4. In addition, the use of DGGE or denaturing gradient gel electrophoresis can be introduced as it can be used as a tool of genetic fingerprinting for a purpose to investigate the diversity of microbial community in specific niches. Using DGGE, a mixture of 16S rRNA fragments with different sequences will resolve into a distinct pattern of bands. PCR and DGGE studies have confirmed that standard culture techniques can be poor indicators of the composition of natural microbial populations.

55

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60

APPENDICES

APPENDIX A

Table : OD at 600nm analysis, cell dry weight and ln X value of strain 2 and strain 5 Time Interval (h) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 OD (600nm) Strain 2 0.028 0.018 0.031 0.027 0.044 0.036 0.048 0.093 0.122 0.230 0.365 0.761 1.305 1.949 5.620 5.960 8.130 8.150 8.100 Strain 5 0.209 0.178 0.224 0.250 0.243 0.378 0.521 0.730 0.816 1.165 1.343 1.511 1.787 7.310 6.980 7.050 8.770 8.400 8.460 X (mg/mL) Strain 2 0.28 0.18 0.31 0.27 0.44 0.36 0.48 0.93 1.22 2.30 3.65 7.61 13.05 19.49 56.20 59.60 81.30 81.50 81.00 Strain 5 2.09 1.78 2.24 2.50 2.43 3.78 5.21 7.30 8.16 11.65 13.43 15.11 17.87 73.10 69.80 70.50 87.70 84.00 84.60 ln X Strain 2 -1.27 -1.71 -1.17 -1.31 -0.82 -1.02 -0.73 -0.07 0.20 0.83 1.29 2.03 2.57 2.97 4.03 4.09 4.39 4.40 4.39 Strain 5 0.74 0.58 0.81 0.92 0.89 1.33 1.65 1.99 2.10 2.46 2.60 2.72 2.88 4.29 4.24 4.26 4.47 4.43 4.44

61 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13 13.5 8.150 8.110 7.690 7.590 6.970 8.600 8.610 8.450 8.440 8.430 8.160 8.100 7.780 7.340 81.50 81.10 76.90 75.90 69.70 86.00 86.10 84.50 84.40 84.30 81.60 81.00 77.80 74.30 4.40 4.40 4.34 4.33 4.24 4.45 4.46 4.44 4.44 4.43 4.40 4.39 4.35 4.31

62 APPENDIX B

Figure OD 600nm analysis plot of strain 2 and strain 5

Figure ln X analysis plot of strain 2

Figure

ln X analysis plot of strain 5

63 APPENDIX C

Table : Value of OD and amount of PHA produce by bacterial strains Bacteria Strain 1 Strain 2 Strain 3 Strain 5 Strain 6 Strain 8 Strain 9 Strain 11 Strain 12 Strain 13 Strain 14 OD Value 10.7455 23.1087 6.01330 23.1496 10.3235 4.36460 6.97910 4.90250 4.41500 3.18360 9.42380 Amount of PHA (mg/L) 0.6934 1.4909 0.3879 1.4935 0.6603 0.2816 0.4503 0.3163 0.2848 0.2054 0.6079

64 APPENDIX D

1.

Beef extract peptone broth:

Composition Beef extract Peptone Potassium nitrate

Amount (g/L) 3.0 5.0 1.0

2. 3.

Reagent A: 0.8% solution of sulfanic acid in 5N acetic acid. Reagent B: 0.5% solution of dimethyl--naphthylamine in 5N acetic acid

*Possible and can be potentially carcinogen.

65 APPENDIX E

Preparation of OF-Glucose Medium

Composition Peptone (pancreatic digest of casein) Sodium chloride di-Potassium hydrogen phosphate anhydrous Agar Bromothymol blue (1.5% w/v stock) Glucose (10% w/v stock)

Amount (g/L) 2.0 5.0 0.3 2.5

Method: 1. All the chemical compositions were mixed in distilled water and stirred it to dissolve it. pH was adjusted to 7.0. 2. 3. 4. 3 mL of bromothymol blue was added to the medium. 50 mL of the medium was autoclaved at 121oC for 15 minutes. 5 mL of filter sterilize glucose was added aseptically from the stock solution of glucose. 5. Later, 5 mL of the medium was dispensed in sterile test tubes.

66 APPENDIX F

Preparation of Triple Sugar Iron (TSI) Agar

Composition Beef extract Yeast extract Peptone Lactose Sucrose Glucose Ferrous sulphate, FeSO4 Sodium thiosulphate, Na2S2O3 Sodium chloride, NaCl Agar Phenol red

Amount (g/L) 3.0 3.0 20.0 10.0 10.0 1.0 0.2 0.2 5.0 12.0 0.024

Method: 1. All the chemical compositions were dissolved into 100 mL of distilled water and pH was adjusted to 7.4. 2. The medium was then poured into test tubes each containing approximately 5 mL to make long slant agar. 3. All the tubes were then autoclaved at 121oC for 15 minutes and cooled to room temperature in slanted position.

67 APPENDIX G

Preparation of Simmons Citrate Agar

Composition Ammonium dehydrogen phosphate Sodium ammonium phosphate Sodium citrate Magnesium sulphate Sodium chloride Alcoholic solution bromothymol blue (1.5% w/v) Agar

Amount (g/L) 1.0 1.0 2.0 0.2 5.0 10.0 20.0

Method: 1. All the chemical composition was dissolved into 1000 mL of distilled water and the pH was adjusted to 7.4. 2. The medium was then poured into test tubes each containing approximately 5 mL to make long slant agar. 3. All the tubes were then autoclaved at 121oC for 15 minutes and cooled to room temperature in slanted position.

68 APPENDIX H

Preparation of Lugols Iodine

Method: 1. 2. 1.0 g of potassium iodide (KI) was dissolved in 100 mL distilled water. Then, 5.0 g of crystal iodine (I2) was added slowly and shake it until dissolved in the mixture. 3. Finally, the solution was filtered and stored in brown bottle

Preparation of Kovacs Reagent

Method: 1. A 10 g of p-dimethylaminoensaldehyde dissolved in 150 mL of pure isoamyl alcohol. 2. A concentrated HCl is then slowly added into the aldehyde-alcohol mixture.

69 APPENDIX I

Preparation of Christensen urea agar slant

Composition Monopotassium Sodium chloride Peptone Glucose Urea Phenol red Agar

Amount (g/L) 2.0 5.0 1.0 1.0 20.0 0.01 15

Method: 1. All the compositions were dissolved in 900 mL of distilled water except urea and autoclaved it at 121oC for 15 minutes. 2. 20 g urea and 1 g glucose were rehydrated in 100 mL distilled water and filter sterilized it using syringe. 3. The urea and glucose solution that have been filtered was added aseptically into the sterile medium. 4. The medium were then poured into test tubes each containing approximately 5 mL and solidify it in slanted position.

70 APPENDIX J

Preparation of Tryptone Broth medium

Composition Tryptophan Yeast extract Sodium chloride di-Sodium hydrogen phosphate

Amount (g/L) 2.0 3.0 5.0 1.0

Method: 1. All the chemical composition above was dissolved in 1000 mL of distilled water and the pH was adjusted to 6.8. 2. The medium were then poured into test tubes each containing approximately 4 mL. 3. The medium was then autoclaved at 121oC for 15 minutes

71 APPENDIX K

Preparation for lipase activity Composition Yeast extract Bacto-Tryptone Methylene blue Agar Tween 80 Amount (g/L) 5.0 10.0 0.15 20.0 1% (v/v)

Method: 1. All the chemical composition above was dissolved in 1000 mL of distilled water and the pH was adjusted to 6.8. 2. The medium was then autoclaved at 121oC for 15 minutes