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
DECLARATION OF THESIS / UNDERGRADUATE PROJECT PAPER AND COPYRIGHT

Author’s 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 :

CONFIDENTIAL RESTRICTED

(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)

OPEN ACCESS

I acknowledged that Universiti Teknologi Malaysia reserves the right as follows : 1. The thesis is the property of Universiti Teknologi Malaysia. 2. The Library of Universiti Teknologi Malaysia has the right to make copies for the purpose of research only. 3. The Library has the right to make copies of the thesis for academic exchange. Certified by :

SIGNATURE

SIGNATURE OF SUPERVISOR

860429-49-6169 (NEW IC NO. /PASSPORT NO.)

DR. ADIBAH YAHYA NAME OF SUPERVISOR

Date : APRIL 2008

Date : APRIL 2008

NOTES :

*

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

i

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

Adibah Yahya Date .” Signature : . : 16th April 2008 Name of Supervisor : Dr..………………………….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).

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. : Himi Husme Bin Abd Majid : 16th April 2008 .. The thesis has not been accepted for any degree and is not concurrently submitted in candidature of any other degree Signature Name Date : ……………………………….

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

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

though only four strains showed enhanced PHA production.4909 mg/L and 1. Cellular characterization showed that strain 2 was a rod shape cell and strain 5 was coccobacilli cell. the colony of strain 5 appeared to be more like fungi and was highly slimy. strain 2 may belonged to Bacillus species and strain 5 can be a Acinetobacter species. In contrast. Results indicated that all bacteria tested were able to produce PHA. . From the biochemical test. the bacteria were screened for their ability to produce PHA in a minimal medium supplemented with glucose as carbon source and grow at 30oC. Strain 2 was observed and showed white flat colony with clear edge zone at each of the colony. 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.vi ABSTRACT Eleven strains of bacteria previously isolated from activated sludge in PHA-producing reactor were used in this study. The 16s rRNA characterization of the bacteria showed that strain 2 also belonged to Bacillus species (82%).4935 mg/L respectively. However. In this study. Morphology characteristic of strain 2 and strain 5 showed that both were clearly distinguish by their shape of colonies. 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.

4935 mg/L setiapnya. 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. Keputusan menunjukkan semua bakteria yang diuji berkebolehan untuk menghasilkan PHA.4909 mg/L dan 1. 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. bakteria tersebut dikesan kebolehannya untuk menghasilkan PHA dalam medium minimum yang ditambah dengan glukosa sebagai sumber karbon dan tumbuh pada 30oC. Dalam kajian ini. Sebaliknya. Pencirian morfologi bagi strain 2 dan strain 5 menunjukkan kedua-duanya dapat dibezakan degan jelas berdasarkan bentuk koloni mereka.vii ABSTRAK Sebelas jenis bakteria yang sebelumnya dipencilkan daripada selut beraktivasi di dalam reaktor yang menghasilkan PHA telah digunakan dalam kajian ini. . Daripada keputusan biokimia. 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). Strain 2 telah diperhatikan dan menunjukkan koloni putih rata dengan zon lutsinar pada setiap koloni. strain 2 dikenalpasti tergolong dalam spesis Bacillus dan strain 5 dari spesis Acinetobacter. Daripada pencirian menggunakan teknik 16S rRNA. Walaupun begitu. strain 2 dikenalpasti tergolong dalam sepsis Bacillus (82%). Walaubagaimanapun. hanya empat jenis bakteria mununjukkan penghasilan PHA yang tinggi.

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 .7 2.1 2.5 2.2 2.3 2.viii CONTENTS CHAPTER TITLE PAGE Title Supervisor’s 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 1 INTRODUCTION 1 2 LITERATURE REVIEW 2.4 2.

3.3 Nutrient Broth Nutrient Agar Mineral Salt Medium 3.1.5.2 3.8 Gelatin liquefaction Test 3.2 Cytochrome oxidase 3.4 3.9 2.3 3.2 Polymerase Chain Reaction 2.1 16S rRNA 2.3 Morphology Characterization of Bacteria Preparation of Inoculums Preparation of Stock Culture 18 18 19 19 20 20 20 21 21 21 22 Media preparation 3.5.10.2 3.7 OF-Glucose Test 3.1.2.5.1.10.5.2.9 Urease Test 23 23 23 23 24 24 25 25 25 26 26 .1.10 Other Types of PHA and Application Characterization and Identification 2.5.3.3 Agarose Gel Electrophoresis 2.4 Gram Staining 2.10.5.1 3.1.2.5 Biochemical Characterization 12 13 13 13 14 15 16 3 MATERIALS AND METHODS 3.1 3.5.2 Experimental Design Microorganisms 3.5.1 3.1.3.1.6 Starch hydrolysis 3.1 Catalase Test 3.1 Biochemical Characterization 3.5 Screening of PHA Producer Characterization and Identification of Potential PHA Producing Bacteria 3.4 Citrate Test 3.5.1.10.5.1.3 Nitrate reduction 3.ix 2.10.5 Triple Sugar Iron Test 3.

14 Voges Proskauer Test 3.3 4.2 Screening of Potential PHA Producer Colony and Cellular Morphologies Characterization 4.5.5.5.2.10 Indole Test 3.6.5 Gram Staining 33 33 35 40 41 46 Biochemical Characterization Bacterial growth analysis PCR Amplication Method for Microbial Identification 4.5.1 4.5.5.11 Motality 3.1 4.1 3.3 Genomic DNA Extraction PCR Amplication of 16s rRNA Gene Fragment Purification and Qualitative PCR Product analysis 4.2.6 Phylogenetic Tree Construction 3.2.1 DNA Extraction 3.2 PCR Amplification of 16S rRNA Fragment 3.2 4.2.6 Analytical Methods 3.4 DNA Sequence analysis 47 47 49 50 51 .1.2.1 4.2.2 Molecular Characterization 3.13 Lipase Test 3.3 Purification of the Amplified 16S rRNA Fragment 3.5.5.1.5.4 Agarose Gel Electrophoresis 3.5.5.6.5.1.1.5 Sequencing of the Amplified 16S rRNA Fragment 3.5.x 3.2.5.2 Determination of PHA Determination of Bacterial Growth 31 31 32 32 32 30 31 29 26 27 27 27 28 28 4 RESULT AND DISCUSSION 4.4 4.5.

2 Conclusion Future Work 54 54 54 REFERENCES 55 APPENDICES 60-71 .4.5.2 ClustalX 51 52 5 CONCLUSION AND FUTURE WORK 5.1 Blastn Analysis 4.xi 4.1 5.5.4.

3 4.2 Cellular characterization of strain 2 and strain 5 4.xii LIST OF TABLES TABLE NO.4 Summary of Biochemical test result Quantitative analysis of mixed culture genomic DNA 41 49 .2 Effect of substrate cost and P(3HB) yield of the production cost of P(3HB) 11 12 29 30 30 2.1 3.2 3.1 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.1 Classification of microbial PHAs according to different criteria 6 2.3 4. TITLE PAGE 2.3 3. 37 40 4.

xiii LIST OF FIGURES FIGURE NO.13 4.14 4.8 4.1 4.2 General structural formula of PHA Principle of biosynthesis of bacteria in bacteria.2 4.5 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 .18 4.11 4. TITLE PAGE 2.15 4.9 4.10 4.16 4. Three relevant phases of biosynthesis of PHA are shown 5 9 18 34 38 38 38 38 38 38 39 39 39 39 39 40 40 42 42 43 43 44 3.1 4.12 4.4 4.17 4.3 4.6 4.7 4.1 2.

80 volts.24 4.28 Agarose gel electrophoresis of the purified PCR products (1% w/v agarose. 45 watts.27 Agarose gel analysis of PCR products (1% w/v agarose. 45 watts.22 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.29 Phylogenetic tree processed and illustrated by Tree View 51 52 50 48 44 45 45 46 46 47 . 80 volts. 60 minutes) 4. 45 watts. 60 minutes) 4. 80 volts.21 4.25 4. 60 minutes) 4.23 4.xiv 4.20 4.

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 C dH2O dsDNA DNA dNTP EDTA EPS H2SO4 HCl g/L kbp µL min h mL M ng/µL 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 A 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 Lugol’s Iodine Preparation of Kovac’s reagent Preparation of Christensen urea agar slant Preparation of Tryptone Broth medium Preparation for lipase activity .

they are completely degraded to water and carbon dioxide (and methane under anaerobic conditions) by microorganisms in various environments such as soil. 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. (PHAs) are polyesters of various hydroxyalkanoates which are synthesized by numerous microorganisms as an energy reserve material. (PHA). Although many bacteria can produce PHA when supplied with the suitable growth condition and carbon substrate. This study was purpose for finding the best bacteria that grow and produce high concentration of polyhydroxyalkanoate. sea and lake water. PHA are same polymer as plastics but it can it degrade more and effectively than normal plastics that are made from petroleum. including plastic waste reduction by developing biodegradable plastic materials. 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.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. not all the bacteria can produce high production of PHA. . Polyhydroxyalkanoates. usually when an essential nutrient such as nitrogen or phosphorus is limited in the presence of excess carbon source.

To characterize selected potential bacteria using biochemical and molecular method . To screen the potential PHA-producing bacteria. 2.2 The objectives of this study are: 1.

Basically. usually under unfavorable growth conditions. limitation of nitrogen. PHAs are stored in the bacterial cytoplasm as inclusion bodies (Lee. sulphur. 2004). 2001). 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 ). ).3 CHAPTER 2 LITERATURE REVIEW 2. magnesium or oxygen in the presence of excess carbon source (Poirier. 1996) and they are synthesized and accumulated intracellularly as distinct granules. . PHAs can be broadly subdivided into three groups based on the number of carbon atoms present in its monomer units (Steinbuchel. 1995). such as feast and famine regime. phosphorus.: (a) Short-chain-length PHAs consisting of 3-5 carbon atoms (PHA SCL ).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.

1. in which the monomer unit is hydroxybutyric acid and the side chain is a methyl group. making these beta or 3-hydroxyalkanoates. and many others can be produced in the laboratory by feeding unusual carbon sources to bacteria. . Most PHAs. As is shown in Figure 2. long molecules made up of many small subunits (monomers) which have been joined together.2 Chemistry of the PHAs Of all the biodegradable plastics being studied.4 2. the monomers are 3-hydroxyalkanoates. these plastics are thus polyesters. the bacterial strain being used and the carbon source utilized to grow the bacteria. PHAs are polymers. The hydroxyl group of one monomer is attached to the carboxyl group of another by an ester bond. these monomers have a hydroxyl group (OH) at the 3rd carbon (what used to be called the beta position). These water-insoluble storage polymers are biodegradable. the polyester linkage creates a molecule which has 3carbon segments separated by oxygen atoms. even what we call PHB. Like all plastics. Other monomer units occur in nature.e. The remainder of the monomer becomes a sidechain off the main backbone of the polymer. are actually copolymers.P. The composition of the polymer synthesized is governed by two main factors. i. 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). exhibit thermoplastic properties and can be produced from renewable carbon sources. and contain some amount of another type of monomer unit. Most of the PHAs encountered in nature are poly(beta-hydroxybutyrate) (PHB). 2007) In the case of PHAs. those that have generated the most interest are the poly(3-hydroxyalkanoates) or PHAs which are made by bacteria. Valappil. Furthermore. (S.

PHBV is a random copolymer.1 General structural formula of polyhydroxyalkanoate (PHA) 2. This remains one of the potential values of PHAs. that by feeding bacteria an appropriate substance. PHB and PHBV have properties similar to polypropylene. Its high degree of crystallinity causes it to crack easily. making processing easier. with its short methyl side chain. Longer side chain polymers are so soft that they are gummy or glue-like. a PHA with specific desirable properties can be produced.3 Physical Properties of PHAs The composition of the PHA has a direct effect on the physical properties of the plastic. . As a result. in. an 8. are elastic. PHB. and bottles made from these polyesters feel just like "normal" plastic. but it produces a much more supple plastic and melts at a lower temperature. it is difficult to use because the temperature at which it melts is very close to the temperature at which it begins to decompose. The flexibility increases with sidechain length throughout the PHA family. largely because of a loss of crystallinity. Polymers composed mostly of hydroxyoctanoate.5 Figure 2.carbon monomer. Industrially. PHBV can still crystallize. meaning that the monomer units do not occur in the chain in any particular order. the PHA used commercially is PHBV. a copolymer of hydroxybutyrate and hydroxyvalerate (5 carbons long). is a very crystalline and very brittle polymer.

. • Monomer size (depending on the number of carbon atoms in an HA monomer • • Short chain-length PHAs (SCL-PHA): contains 3-5 carbon atoms.e Poly(3-hydroxybutyrate) P(3HB).e P(3HB-co-3MP).1 : Classification of microbial PHAs according to different criteria (Luengo. classification according to the monomer size. PHAs containing both aliphatic and aromatic fatty acids. and the type of polymer is the most common and will be described in detail here.6 PHAs can be classified into various groups according to different criteria (Table 2. i. A homopolymer is produced when single monomeric units are linked together. Table 2. Medium chain-length PHAs (MCL-PHA): contains 614 carbon atoms.e P(3HB-co-4HB). i. 2003) • Natural PHAs: produced naturally by microorganisms from general substrates. i. PHAs containing aromatic fatty acids. a copolymer is formed. 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)].1). Heteropolymer: When two or more different monomeric units are linked together. 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. i.e P(3HB). • Chemical nature of the monomers • • PHAs containing aliphatic fatty acids.e P(3HB). which refers to the number of carbon atoms in the HA monomer. Among them. i.

discrete. 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. PHAs are one such storage compound. PHAs are usually produced when carbon sources are in excess. PHAs are an excellent storage compound because their presence in the cytoplasm. Both stains however can stain lipid bodies. Nile Blue A is a more specific dye than Sudan Black B as it does not stain glycogen and polyphosphate. The number and sizes of granules per cell differ depending on the PHA-producer microorganisms and their growth stage. The carbon sources are assimilated. exhibiting a strong orange fluorescence. PHA granules can be observed as refractile granules under phase contrast light microscope. 1970) and more specifically by Nile Blue A.2-0. 2000). even in large quantities does not disturb the osmotic pressure of the cell.4 The Biology of PHA In nature. When thin sections of cells containing PHAs are viewed under transmission electron microscope.1. converted into hydroxyalkanoate (HA) compounds and finally polymerized into high molecular weight PHAs and stored as water insoluble granules in the cell cytoplasm. the granules appear as electron transparent. spherical particles with clear boundaries. In Wautersia eutropha (formerly known as Alcaligenes eutrophus). Numerous bacteria can synthesize and accumulate PHAs as carbon and energy storage materials or as a sink for redundant . 2.5 Biosynthesis of PHA Polyhydroxyalkanoates are polyesters of hydroxyalkanoates (HAs) having the general structural formula shown in Figure 2.7 2. 8-13 granules per cell with sizes ranging from 0.5 m were detected (Byrom. 1994). PHA granules could be stained with Sudan Black (Schlegel.

Regarding the application of precursor substrates. At present it cannot generally be excluded that the PHA synthases also use other thioesters of HA as substrates. since during this phase the carbon source is converted into a suitable substrate for the PHA synthase. for example. 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). anabolic or catabolic reactions. If the carbon source is not a precursor substrate. 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. which is provided as a carbon source to the cells. Three metabolic phases of the biosynthesis of PHA in bacteria can be distinguished (Figure 1. Phase II is of most importance. First. .8 reducing power under the condition of limiting nutrients in the presence of excess carbon (Steinbuchel. the most simple type of reaction is the conversion of a HA. by a thiokinase or a coenzyme A transferase into the corresponding HA-coenzyme A thioester. Third.3). uses these thioesters as substrates and catalyzes the formation of the ester bond with the concomitant release of coenzyme A. which is the key enzyme of PHA biosynthesis. such as. convert the compound into a hydroxyacyl coenzyme A thioester which is a substrate of the PHA synthase. the conversion of 4HB into 4-hydroxybutyryl-coenzyme A. 1995). 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. Second. 1991).

and the maximum extent of polymer accumulation. It is. . 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. important to develop cultivation strategies that can simulate these conditions for the efficient production of PHA.PHA material. Selection of a microorganism for the industrial production of PHA should be based on several factors including the cell’s ability to utilize an inexpensive carbon source. Three relevant phases of biosynthesis of PHA are shown. Three relevant phases of biosynthesis of PHA are shown 2. growth rate. (Steinbiichel. The yield of PHA on carbon source is important not to waste substrate to non.6 Production of PHA Figure 2. therefore. 1990). 1995).9 Figure 2. Recovery of PHA should also be considered because it significantly affects the overall economics. or oxygen in the presence of excess carbon (Anderson.2: Principle of biosynthesis of bacteria in bacteria. magnesium. phosphorus. polymer synthesis rate. 1992).2 Principle of biosynthesis of bacteria in bacteria. 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. In most bacteria PHAs are synthesized and intracellularly accumulated under unfavorable growth conditions such as limitation of nitrogen.

cells containing PHAs are separated by conventional procedures such as centrifugation. The first method that has most often been used involves extraction of P(3HB) from biomass with solvent. The large amount of solvent required makes this method economically unattractive. or vortexing. propylene carbonate. and thus protects polymer from degradation. and can be predicted from the physiology and biochemistry involved in the PHA synthesis. A . 2. A number of different methods have been developed for the recovery of PHA. even after the recycling of the solvent (Holmes.7 Recovery of PHA Following the fermentation. filtration. Several other methods that have been developed involve the use of sodium hypochlorite for the differential digestion of non-PHA cellular materials (Berger. the carbon source contributes most significantly to the overall substrate cost in PHA production. The efficiency of substrate conversion is important. Due to the high viscosity of even dilute PHA solutions. The solvents employed include chloroform.8 Carbon substrate and yield Excluding the recovery process. Normally. It was suggested that chloroform immediately dissolves the isolated P(3HB) by hypochlorite. 1994). cells are disrupted to recover the polymers. polymer purity of greater than 95% is obtained by hypochlorite treatment. The use of sodium hypochlorite together with chloroform significantly reduced degradation of PHA (Hahn. Although this method is effective in the digestion of non-PHA cellular materials. After the biomass harvesting. 1989). it causes severe degradation of P(3HB) resulting in a 50% reduction in the molecular weight.10 2. and dichloroethane. Among the various nutrients in the fermentation medium. 1994). 1994). methylene chloride. about 20 portion of solvent are required to extract 1 portion of polymer (Byrom. the economics of PHA production are largely determined by the substrate cost and PHA yield.

11 number of carbon sources. The theoretical yield (the yield based on the reaction stoichiometry) of P(3HB) has been estimated for several of these carbon substrates (Yamane. 1993).72 0.56 1. has been taken into account in this analysis.290 0. including carbohydrates.42 1.40 0.595 0. oils. cheese whey.180 0.33 Substrate cost 1.38 0.071 Substrate Glucose Sucrose Methanol Acetic acid Ethanol Cane Molasses Cheese Whey P(3HB) yield 0. which are used as cofactor for PHA synthesis. It was also suggested that the overall yield.50 0. which is the yield in actual fermentation. Table 2.2 : Effect of substrate cost and P(3HB) yield of the production cost of P(3HB) Approximate price (US$/kg) 0.2 summarizes the cost of carbon substrate based on the theoretical yield. cellulose and hemicellulose can be excellent substrates to several bacteria utilizing them.52 0. acids and hydrocarbons.42 0.38 0. alcohols.220 0. Regeneration of nicotinamide nucleotides. 1993).493 0. would be roughly proportional to the theoretical yield and PHA content. plant oils and hydrolysates of starch (corn and tapioca).502 0. Table 2.22 . crude carbon substrates such as the cane and beet molasses. Because of their low price. can be used by various bacteria (Yamane.43 0.30 0.00 0.

It was used for tissue engineered heart valve scaffold and viable ovine blood vessels (Chen and Wu. The copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). insecticides. disposable food service ware and moulded products such as bottles and razors and also be used for biomedical applications (Lee. P(3HB). The material properties and hence the application of the PHAs vary depending on the monomers composition. is the most well known and well characterized PHA. However. is more flexible and tougher than the P(3HB).3: The possible application of PHA • • • • • • • Packaging films. or feminine hygiene products Surgical pins. P(4HB).3.9 Other types of PHA and application Beside PHA. 2003). can be produced by Comamonas acidovorans with a controlled degradation rate (Saito and Doi. 2003). there are many types of PHA. compost bags. diapers. Polyhydroxybutyrate. It can be used to make various products. P(3HB-co-3HV). has been found to be useful in the biomedical applications (Martin and Williams. 1996). The P(4HB). a high molecular weight copolymer of 3HB and 4HB [P(3HB-co-4HB)] containing 0–100 mol% of 4HB. including films. Also. or fertilizers Disposable items such as razors. medicines. making them ideal candidates for biomedical applications such as tissue engineering (Martin and Williams. coated paper. and swabs Wound dressing . herbicides. 1994). there have recently discovered 4-hydroxybutyrate. industrial applications of P(3HB) have been hampered knowing to its low thermal stability and excessive brittleness upon storage (Lee. bags and containers Biodegradable carrier for long term dosage of drugs. board. Table 2. staples. 2005). sutures.12 2. utensils. Besides that. Other application of PHA are shown in Table 2. 1996).

The primers are one component of the reaction mixture. meaning that sequence differences appear to provide a relative measure of the time elapsed since the organisms diverged from common ancestor (Willey. DNA sequences are viewed as evolutionary chronometers.13 2. which also contains the target DNA.10. the temperature is lowered so that the . 2008). These synthetic oligonucleotides are usually about 20 nucleotides long and serve as DNA primer DNA synthesis. These trees are somewhat like a family tree. and metabolic capabilities to group organisms. a thermostable DNA polymerase. the target DNA containing the sequence to be amplified is heat denatured to make it single stranded. 2008). Next. Newer molecular techniques such as DNA sequencing give a greater insight into the evolutionary relatedness of microorganisms.2 Polymerase Chain Reaction The first step is to synthesize DNA fragment with sequence identical to those flanking the targeted sequence. Individual species are represented as nodes.10 Characterization and identification of bacteria 2. PCR requires a series of repeated reactions. tracing the evolutionary heritage of organisms.1 16S rRNA Prokaryotic classification has historically relied on phenotypic attributes such as size and shape.10. DNA sequencing enables one to more accurately construct a phylogenetic tree. 2. In the first step. Each cycle has three step that are precisely executed in a machine called thermocycler (Willey. called cycles. staining characteristics. and each of the four deoxyribonucleoside triphosphates (dNTPs). Each line or branch of the tree represents the evolutionary distance between two species. This is accomplished with a DNA synthesizer.

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 fragment’s 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 Gram’s 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

1 Morphology characterization of bacteria Pure cultures of bacteria were streaked onto Nutrient agar medium (Section 3. streaked onto a separated Nutrient agar medium and allowed to grow at 30oC for 24 h. The purity of each bacterial culture was monitored by streaking onto separate nutrient agar medium (Section 3.1) and incubated at 30oC for 24 h without shaking. strain 3. strain 12.2 Microorganisms Eleven strains of bacteria coded strain 1.2) and incubated at 30oC for 24 h. strain 11. Faculty of Science. strain 6. strain 13 and strain 14 were obtained from the culture collection of Research Laboratory 2.2) and incubated for 24 h at the same temperature.3. strain 5. . whereas those observed with two or more types of colonies are subjected to culture purification via single colony isolation method. 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. Skudai. 3. 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. Universiti Teknologi Malaysia. The bacterial colonies grown on the solid medium were then observed under stereo microscope (Leica model CME microscope). strain 2. strain 9.3.3. A single colony of different morphologies were aseptically selected. Repeated single colony isolation was carried out until the culture purity was ensured. Department of Biology.2. strain 8. Culture observed with colonies of the same morphology on nutrient agar is considered pure culture.19 3. Johor.

3. 3.20 3.1) at 30oC for 24 h.8 mL of the fresh cultures were then transferred into a separate sterile Eppendorf tube and added with 0. 200rpm for 24 h. The cultures were then frozen in liquid nitrogen prior to place at -80oC freezer for long term storage.8 were used as inoculums. Cultures with the absorbance values ranging from 0.3.3.2.1). The culture’s turbidity was measured using a bench top spectrophotometer (Jenway 6300) at the wavelength of 600nm.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. .3.3 Media preparation Solid and liquid media used in this study are of enriched and defined types.1 to 3.2 Preparation of inoculums The pure bacterial strains were streaked onto the Nutrient agar medium (Section 3. A 0.3.2 mL glycerol to a final volume of 15% v/v.3. The bacteria were allowed to grow in a shaking incubator at 30oC.7-0.3. All media are prepared as described in section 3.2).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. They were mixed and sterilized via autoclaving at 121oC for 20 min. 2. Nutrient broth consists of peptone. An 8 g/L of Nutrient broth powder was added into a 1L Schott bottle containing 1L of distilled water.3. sodium chloride as the carbon source of chloride ion and agar as gelling agent.3. The broth was left to cool to 50oC prior to pour into sterile universal bottle. All plates were sealed with parafilm in order to avoid contamination during storage.5 (NH4)2SO4.12H2O.2 Nutrient Agar Nutrient agar is commercially obtained which consisted of peptone and beef extract as the source of carbon.1 Nutrient Broth This is an enrichment medium used to grow the bacteria when preparing the inoculums.3 Mineral Salt Medium For PHA production from all the bacterial strain. 0. The mineral salt media consisted of (g/L) 0.7H2O. the medium was used for the preparation of bacterial inoculums. Nutrient broth has been a commonly used medium to grow heterotrophic bacteria. A 20. 3. The medium were left to cool at 50oC prior to pour into sterile Petri plates.21 3. the strain was grown in mineral salt media containing 10 g/L glucose and microelement. The agar was left to solidify at room temperature. 9. The plates can be directly used or stored at 4oC for later used. minerals and vitamins which are important to support bacterial growth.65 Na2HPO4.0 g/L of the nutrient powder was added into a 1L Schott bottle containing 1L of distilled water. The universal bottle can be directly used or stored at 4oC for subsequent use. In this study.65 .4 MgSO4. meat extract and distilled water. They were mixed and sterilized via autoclaving at 121oC for 20 min.3.

4 Screening of PHA Producer This screening method was used for detection of PHA production after cultivation in mineral salt media.7H2O in 0. The supernatant containing chloroform is evaporated and 5 mL of concentrated H2SO4 were added. wash with water and then wash with alcohol and acetone.1 ZnSO4.05 MnCl2. 1mL from the solution containing 20g FeCl3.7H2O. 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.22 KH2PO4 in distilled water.5M HCl was added to the mineral salt media.H2O. After 1 h at 37oC. the lipid granule were centrifuged. The purpose of this is to check or screen which bacterial strain can produce high production of PHA. 10g CaCl2. The polymer was dissolved with 5 mL of chloroform and left overnight at 28oC on a shaker at 150rpm. The solution is cooled to room temperature and the samples are transferred to silica cuvette and the amount of PHA was determined at 235nm. Then the contents were centrifuged at 4000 x g for 30 minutes. The supernatant was taken and transferred into clean test tube. Before continuing the screening. The MgSO4. The cell pellet or paste was suspended in a volume of commercial sodium hypochlorite solution (Clorox) equal to original volume of medium.6H2O. 0.03 CuSO4. 0.5H2O. 3. All the content was autoclaved at 121oC for 20 min except for MgSO4.7H2O was filter sterilized and added to the autoclave mineral salt media.4H2O and 0. For the microelement. . The tube is capped and heated for 10 min at 100oC in water bath.

3. The drops of hydrogen peroxide were observed to see if bubbles were involved.5 3.1.1 Characterization and identification of potential PHA producing bacteria Biochemical Characterization Enzymatic activities are widely used to differentiate bacteria. Moreover. OX+ normally means that the bacterium contains cytochrome c oxidase and can therefore utilize oxygen for energy production with an electron transfer chain. Even closely related bacteria can usually be separated into distinct species by subjecting them to biochemical tests.1 Catalase Test This test is particularly useful in distinguishing staphylococci and micrococci. The production of gaseous bubbles indicates the presence of catalase.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-). 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. such as one to determine their ability to ferment an assortment of selected carbohydrates. A loopful of bacterial growth was aseptically transferred from agar medium and smear it . 3. biochemical tests can provide insight into a species’ niche in the ecosystem.5. A drop of hydrogen peroxide was pipette onto the mass of bacterial cells.1.5. which are catalase-positive. which are catalasenegative The test was done aseptically by picked up bacterial cell from colony of slant growth with an inoculating loop.5. from streptococci and enterococci.23 3.

Simmons citrate agar contains sodium citrate as the sole source of carbon. The development of violet or purple colour after observing the filter paper within 10 seconds indicates a positive test.5.24 on the moisten paper. All the tubes were then incubate at 37oC for 4 days and observed any changes occurred. which creates an alkaline environment in the medium. bacterial culture was streak onto citrate slant agar (Appendix G) and was labelled accordingly to each strain.1. Any immediate red colour demonstrates the presence of nitrite and is indicative of reduction of nitrate and nitrite. 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.4 Citrate Test Simmons citrate agar tests the ability of organisms to utilize citrate as a carbon source. 2-3 drops of Reagent A (Appendix D) and 2-3 drops of Reagent B (Appendix D) were added to each tube. 3. Organisms which can utilize citrate as their sole carbon source use the enzyme citrase or citrate-permease to transport the citrate into the cell.5. ammonium dihydrogen phosphate as the sole source of nitrogen and other nutrients. . The test was perform by inoculate bacterial cultures onto tubes containing beef extract-peptone broth (Appendix D). 3.3 Nitrate reduction A nitrate test is a chemical test used to determine the presence of nitrate ion in solution.1. These organisms also convert the ammonium dihydrogen phosphate to ammonia and ammonium hydroxide. Using a sterile loop.

6 Starch hydrolysis The test involves the breakdown of starch into maltose.5.5. The plates contained sterile nutrient agar supplemented with 0. ferrous sulfate.7 OF-Glucose Test OF medium is a nutrient semisolid agar containing high concentration of carbohydrate and low concentration of peptone. sucrose. A sterile inoculating loop with bacterial culture was streak onto TSI slant agar (Appendix F). All the plates were incubated at 37oC for 1 to 5 days. It is used to differentiate enteric based on the ability to reduce sulfur and ferment carbohydrates. and the pH indicator phenol red.1.1.5. 2ml of sterile mineral oil was poured over one of the inoculating tube and replaced the . 3.5 Triple Sugar Iron Test Triple sugar iron agar (TSI) is a differential medium that contains lactose. The peptone will support growth of bacteria that do not use carbohydrate. After incubation.1. 3. The tubes were labelled carefully and incubate it at 37oC for 24 h. Any clear are indicates the hydrolysis of starch and unchanged starch will stain dark blue.25 3. The test start with using a sterile needle and the bacterial culture was inoculated each with two tubes of OF-glucose media (Appendix E).2% (w/v) soluble starch. another bacterial culture was stabbed onto another TSI slant agar. a small amount of glucose (dextrose). Using the same culture. all the bacterial strain was inoculated by making a single streak down the middle of the plates. Firstly. the plates were flooded with Lugol’s iodine (Appendix H).

10 Indole Test Tryptone broth (Appendix 10) tubes were inoculated with bacterial strain. 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.1.1. All the tube then incubated at 37oC for 24 to 48 hours.1. soybeans. and other plant seeds.5.5. It is found in large quantities in jack beans.9 Urease Test Urease is an enzyme that catalyzes the hydrolysis of urea. The tube was labelled as anaerobic and the other (not added with mineral oil) as aerobic tube. 3. All the tubes were incubated at 37oC for 15 days. Bacterial culture was inoculated in the nutrient broth-gelatin tubes and all the tubes was labelled and incubated at 37oC for 10-14 days. Lack of gelatin hydrolysis will result in liquid consistency of the medium.5. the appearance of a red violet colour indicates a positive test while a yellow orange colour indicates negative result.26 cap. After the incubation. 10 drops of Kovac’s reagent (Appendix . The tubes were placed in a refrigerator for 30-60 minutes after incubation. 3. Both tubes were incubated at 37oC for 1 or 2 days.8 Gelatin liquefaction Test Many microorganisms produce gelatinase which catalyzes the hydrolysis of the collagen. 3. forming ammonia and carbon dioxide.

1.0g). In this test.1. 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. a natural product formed from pyruvic acid in the course of glucose fermentation. The media was autoclaving at 121oC for 15 minutes.0g) in 1L of distilled water. .14 Voges Proskauer Test The principle for this test is to detect the production of acetylmethylcarbinol.5. NaCl (5. Positive result show pink colour after added α-napthol. peptone (10.0g).5.1. The medium used for the test is Spirit Blue agar (Appendix K).0g) and Agar (4. and 1 mL of 40% KOH.11 Motality Motality test are used to identify the organism or bacteria that are able to move. positive test indicates the organism is motile which cause turbidity in the medium.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. 3.13 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 media contain beef extract (3.5.

5.1 DNA Extraction A 5 ml of overnight culture was added into a centrifuged tube.5M EDTA.000 rpm for 3 minutes.2. the pellet was resuspended in 480µl of 0. 120µl of lysozyme was added gently mixing by inverting the centrifuge tube and later incubated it at 37oC for 1 hour.000 rpm for 5 minutes. 3µl of RNase solution was added to the cell lysate and let it mix by inverting the tube for several time. The tube was centrifuged at 13. After 1 hour. The success of genomic DNA isolation was determined by agarose gel electrophoresis analysis. Further incubation was perform for the culture on ice for 5 minutes and centrifuged at 13. The supernatant was transferred into a fresh centrifuged tube which contains 600µl isopropanol (at room temperature) and gently mix it by inverting the tube.28 3.000 rpm for 3 minutes. 100µl of DNA rehydration solution was added to the tube and incubated overnight at 4oC. 600µl of 70% (v/v) ethanol was added and gently mix it to wash the DNA pellet and followed by centrifuged the tube at 13. The supernatant was removed and 600µl of nucleic lysis solution was added and mix gently until the cell resuspended. The supernatant was carefully drain and air dried for 10-15 minutes. The tube was centrifuged at 13. The suspension then incubated at 80oC for 5 minutes to lyse the cell and let it cool to room temperature.000 rpm for 3 minutes.5. A 200µl of protein precipitation solution was added to the RNase-treated cell lysate and vortexing it at high speed. The supernatant was carefully poured and drain the tube on clean absorbent paper.2 Molecular Characterization 3.000 for 3 minutes. Then. . The supernatant were removed and discarded. the culture was centrifudge at 13. The mixture was then incubated at 37oC for 30 minutes and let it cool to room temperature.

genomic DNA template was added accordingly to the concentration of the genomic DNA. . forward and reverse primer.AAG GAG GTG ATC CAG CCG CA -3’ Mixture to run the PCR contains PCR Master Mix (Promega).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).AGA GTT TGA TCC TGG CTC AG -3’ 5’. genomic DNA template and nuclease free water. primer.2.1.5oC Table 3. annealing temperature (Ta) of the primer must be known.5 o C = 2(A+T) + 4(G+C) . the annealing temperature starts at 5oC below the calculated temperature of primer melting point (Tm).29 3. Usually. To start the PCR. PCR Master Mix and nuclease free water was added to the total of 50 µL of all the mixture. Ta = T m . To prepare. The sequences of respective primer are shown in table 3.5.1 : Sequences of eubacterial 16S rDNA universal primers Primers pA pH Sequence 5’.

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 3.5.3 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 manufacturer’s instructions.0 µM 0.1 – 1. salts. agarose.2 : PCR reaction mixtures Reagents DNA Template Forward Primer Reverse Primer 2X PCR Master Mix Nuclease Free Water Total Volumes (µL) Strain 2 1 1 1 25 22 50 Strain 5 3 1 1 25 20 50 < 250 ng 0. and other impurities from the DNA samples. ethedium bromide.1 – 1. The purpose for doing the purification is to remove enzymes.0 µM 1X Final Concentration Table 3.2.30 Table 3. Purication method can be done by using two methods. gel dissolving and direct PCR product solution. primers. nucleotides. . mineral oil.

Carefully. Selangor. were sent to 1st BASE Laboratory Sdn.5 Sequencing of the Amplified 16S rDNA Fragment The PCR product that have been purified.2. Then. 3. Firstly.31 3. Selangor. were analyzed using online software BLASTn (Basic Local Alignment Search Tool of nucleotide).. Bhd.5.4 Agarose Gel Electrophoresis First step in doing agarose gel electrophoresis is to prepare the agarose gel. the combs were removed by pulling them upwards firmly and smoothly in a continuous motion. A 10kb mass ruler DNA marker was used as standard for determining the size of DNA fragment. The suspension was heated in a microwave until dissolved. Both forward and reverse primer was also included as sequencing probe. The solution was left to cool at about 40oC and ethidium bromide was added into the molten agarose solution. Bhd. The gel was run at 80V for approximately 1 hour and observed.2. After finished preparing the gel.. The sequence match obtain from the BLASTn are subjected to ClustalX .2.5. 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. The mixture was loaded into the well. 1g of agarose was weight out and added into 50ml 1XTAE buffer. 1µl of loading dye was added and mix it by flicking the tube several times. 3. The electrophoresis chamber was closed with lid and connects all power cable according to the colour code. a 5µl of the DNA sample was aliquot into a microcentrifuged tube.6 Phylogenetic Tree Construction Sequence result obtained from 1st BASE Laboratory Sdn. the next step is to prepare the DNA sample for electrophoresis.5.

. the solution (Section 3. 3. It calculates the best match for selected sequence and lines them up so that the identities.32 program to align the multiple sequence. The solution was diluted to the dilution factor of 10. 1 mL from each flask was transferred to cleaned cuvettes to check the optical densities by using spectrophotometer at 600nm. This curve is affected by environmental and nutritional factors.6.6 3. the lag phase. 3. Before the determination. the initial solution contain concentrated sulphuric acid and cannot be read by the UV-Spectrophotometer.6. Evolutionary relationships can be seen via tree view application. 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 absorbance of the UV-Spectrophotometer was set to 235nm and all the absorbance reading of each samples was recorded and check. the log phase. similarities and differences can be seen. This is because.4) was diluted with sterile distiller water.2 Determination of Bacterial Growth The normal bacterial growth curve has four phases.1 Analytical Methods Determination of PHA PHA that was extracted from cell was determined by using UVSpectrophotometer. The time interval to measure the optical densities is every 30 minutes. the stationary phase and death phase.

Because the extraction used solution that are dangerous which are some volatile and corrosive. 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. Before the harvesting of PHA. all the centrifuged tubes were washed with ethanol and hot chloroform. ethanol. The medium used to grow the bacterial strains can affect the production of PHA from bacteria. Referring to Wennan He. During the extraction of the PHA from the cultivated bacterial strains. (1998).CHAPTER 4 RESULTS AND DISCUSSIONS 4. Some solution such as hot chloroform. there are some precaution procedures that must be taken seriously. PHA was produced by the bacteria intracellulaly. Different kinds of smell were also produced. the use of lab coat. some of the bacterial strain produced slimy and foam. The productions of PHA are depended on the environmental factor. By using mineral salt media. During the cultivation of the bacteria. the PHA production was successfully produced from the bacteria. latex glove and mouth mask must be used. .1 Screening of potential PHA producer Some bacteria have been identified that have the ability to produce PHA. growth condition and the nutrient available. The mineral salt media are supplemented with glucose as the carbon source. and concentrated sulfuric acid must be handling with care. PHA production can be enhanced by using mineral salt media. The PHA produced in the cell were harvested by using dispersion of chloroform and aqueous sodium hypochloride.

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. Figure 4. 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.1 Amount of PHA produce by bacterial strains . So.

Strain 8. 2. Universiti Teknologi Malaysia. 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. .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. glucose was also found to be the favorable substrate (carbon source) for PHA production. Skudai.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. Faculty of Science. strain 12 and strain 13 showed low production of PHA. 1960). Department of Biology. 5 and 6 were among the potential PHA producers. results showed in Figure 4.35 The amount of PHA extracted was determined as the following (Slepecky and Law. Based on the report by Wennan He. All the strains of pure colony show different characteristic in morphologies and sizes.55 x 10-4 x 1 x c x DF c = OD x DF 1. (1998). A = kbc OD = 1. Johor. However. two strains which are strain 2 and strain 5 showed highest production of PHA compared to other strains. This maybe due to the limited amount of cell or they may favor other environmental factor and other carbon source besides glucose.1 indicated that strains 1. 4.

. strain 8 (Figure 4. strain 6. strain 8 and strain 13). for strain 8 and strain 13. For example. These attachments are due to the present of glycocalyx. plant roots and even on bacteria. the glycocalyx can also protect a cell against dehydration. strain 11 and strain 12) and pink (strain 14). 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.11). Most of the strains appeared to have rough surface due to the characteristic of the strains and colony growth. yellow (strain 9.7) and strain 13 (Figure 4. Strain 2 and strain 3 show similarity in the shape of colony. strain 2. strain 5.36 Some of the strains grow very turbid and produce slime which is called EPS. Many of the strains show same pigmentation colour which is white (strain 1. However. EPS is an extracellular polysaccharide which enables a bacterium to survive by attaching to various surfaces in its natural environment in order to survive. bacteria can grow on diverse surfaces such as rocks. strain 3. strain 2 grow faster and have a clear zone at the end of the colony and differ to strain 3. However. Through attachment. Besides giving attachment.

37 Table 4. Colony surface Optical Characterization Strain Forms Elevation Margins Pigmentation 1 filamentous umbonate filamentous rough white opaque 2 circular raised undulate rough white translucent 3 circular raised undulate rough white opaque 5 circular convex entire rough milky white opaque 6 circular effuse entire rough white translucent 8 irregular umbonate entire smooth milky white opaque 9 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 .1 : Colony morphologies of pure colony on Agar plate culture.

38 Figure 4.6 Colony morphology Strain 6 Figure 4.7 Colony morphology Strain 8 .4 Colony morphology Strain 3 Figure 4.2 Colony morphology Strain 1 Figure 4.3 Colony morphology Strain 2 Figure 4.5 Colony morphology Strain 5 Figure 4.

12 Colony morphology Strain 14 .39 Figure 4.11 Colony morphology Strain 13 Figure 4.9 Colony morphology Strain 11 Figure 4.8 Colony morphology Strain 9 Figure 4.10 Colony morphology Strain 12 Figure 4.

14 Cellular morphology of strain 5 . The cellular characteristics of the strains are shown in Table 4. Strain 2 and strain 5 was selected to be characterize because its ability to produce high amount of PHA.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. It can be possibly related to Bacillus species. Its cellular arrangement is similar to branching coccobacilli which is oval and cocci in shape.2. strain 5 appeared to be gram negative.1 Gram Staining Gram staining was done to further characterize the pure colony of the bacterial strains to observe their cellular morphology and arrangement.40 4. Figure 4.2: Cellular characterization of strain 2 and strain 5 Strain 2 Figure 4. Table 4. Its cellular arrangement is rod shape.27 5 Figure 4. Meanwhile.13 Cellular morphology of strain 2 Figure 4. It can be possibly related under phylum Acinetobacter.2. the gram staining results revealed that strain 2 appeared to be gram positive.

From the catalase test. gram staining. urease. lipase.3 Biochemical Characterization Biochemical test are used to determine and to differentiated the bacteria based on their properties. Table 4. strain 2 show no reaction which give negative result and .3 summaries the biochemical test that have been done on strain 2 and strain 5. OF-Glucose.3. The biochemical tests were catalase. Voges-proskauer and motility test.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. starch hydrolysis. indole. cytochrome oxidase. MacConkey agar. nitrate reduction. TSI. citrate. gelatin liquefaction.41 4. The results are summarized in the Table 4.

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. The presence of nitrogen gas release from the strains is showed by red color formation.16). Most aerobic microorganisms possess catalase. The nitrate may be further reduced to nitrogen gas (Jean F. In anaerobic respiration using nitrate as the terminal electron acceptor. The strain can use nitrate as the terminal electron acceptor in place of oxygen during respiratory metabolism. Figure 4. Catalase is an enzyme that catalyzes the decomposition of hydrogen peroxide (H2O2) to water and gaseous oxygen. 1980). Positive result for citrate test is shown by turbidity and blue color changes from green color medium. the pathway is called anaerobic respiration.42 strain 5 show reaction with production of bubbles which give positive result. 1980).15.16 Nitrate reduction test of Strain 5 Strain 2 and strain 5 did not show any positive result to the citrate test. When this occurs. 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. For the nitrate reduction test. This is because the strains do not utilize citrate as the sole carbon source of carbon. which changes the color from green to blue (Jean F. nitratase catalyzes the reduction of nitrate to nitrite. The organisms that utilize citrate and produce an alkaline reaction as indicated by the bromothymol blue. 1980). both of strain 2 and strain 5 showed positive result (Figure 4. Figure 4. .15 Nitrate reduction test of Strain 2 Figure 4.

1980). Figure 4. show negative results for starch hydrolysis test and this showed that it cannot breakdown the starch. along with the possible hydrogen sulfide (H2S) production.17 Starch hydrolysis test on strain 2 Figure 4.43 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. By the fermentation process. Positive results on the medium are shown by purple-blue with colorless area around growth of bacteria (Jean F. forming a polymer of many units united by α-glucosidic linkages. Fermentation is an aerobic process and bacterial fermenters of a carbohydrate are usually facultative anaerobes. a . α-D-glucose. Starch hydrolysis test is to check the presence of extracellular amylolitic enzymes that breakdown starch. Starch is a homopolysaccharide. Strain 2 and strain 5 show positive results for the fermentation of glucose and lactose (Jean F.18 Starch hydrolysis test on strain 5 Oxidative or fermentative organism can be determined by using Rudolph Hugh and Einar Leifson’s 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.18) showed positive result and was able to breakdown the starch. 1980).17). Strain 2 (Figure 4. a condensation product of many monomers of a single type of monosaccharide. Strain 5 (Figure 4. 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. with or without the production of gas.

1980). . Microorganisms that produce this enzyme are able to detoxify a waste product and derive metabolic energy from its utilization Figure 4. Yellow color formation on both medium confirmed the results. Lipase test are used to determine whether a bacterium produces a lipase that will hydrolyze a neutral fat to fatty acid and glycerol. Strain 2 and strain 5 are both fermentative bacteria by the result of OF-Glucose test.44 carbohydrate is metabolized and split into two triose carbon molecules (Jean F. gave negative results howeverpositive for strain 5 (Figure 4. Gelatin liquefaction test showed positive result for both strain 2 and strain 5. wioth red color changes of the medium. Urease test on strain 2 (Figure 4.20 Positive result of urease test of strain 5 Strain 2 and strain 5 showed positive result of lipase activity.19 Negative result of urease test of strain 2 Figure 4.20). catalyze the breakdown of urea into ammonia and carbon dioxide. The hydrolysis of gelatin produces soluble carbohydrates that are readily metabolized as a source of carbon source. .19). The enzyme urease. The strains possess gelatinase which is produced to catalyze the hydrolysis of the protein gelatin (collagen).

From pyrivic acid there are many pathways a bacterium may follow. The production of acetoin is one pathway for glucose degradation occurring in bacteria.24). Strain 2 and strain 5 are nonmotile bacteria from the motality test. Nonmotile organisms lack flagella. from glucose fermentation. a few coccal forms are motile. Both strain 2 and strain 5 lacks this ability. The purpose for motality test is to determine if an organism is motile or nonmotile. However. The ability to hydrolyze tryptophan to indole is a characteristic of strain enteric bacteria that possess the enzyme trytophanase. Motile bacteria may contain a single flagellum or many flagella. Strain 5 (Figure 4. 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.22 Gel liquefaction test of strain 5 Voges Proskauer test is used to determine the ability of organisms to produce a neutral end product.45 Figure 4. pyruvic acid and water. is gram negative bacteria by means of growth on MacConkey agar and gram staining. Bacteria are motile by means of flagella. Flagella occur primarily among the bacilli. . strain 2 and strain 5 did not have this ability and thus give negative result in the indole test. Trytophanase catalyze the hydrolysis of tryptophan with the production of indole. acetylmethylcarbinol (acetoin). However. Glucose is metabolized to pyruvic acid which is the key intermediate in glycolysis.21 Gel liquefaction test of strain 2 Figure 4.

the availabilty of nutient and biomass accumulated end or less when the phase started to enter stationary phase.46 Figure 4.4 Bacterial growth analysis Bacterial growth refers to an increase in bacterial numbers. not an increase in the size of the individual cells. 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. Its showed that strain 2 are related to genus Bacillus species and strain 5 are related under phylum Acinetobacter. the log phase. PHA probably being produce at the log phase and as time passed. 4. it proceeds exponentially with one cell dividing to form two.23 No growth on MacConkey agar of strain 2 Figure 4. Referring to the cell dry weight analysis.25. Strain 2 and strain 5 shows normal growth curve of bacteria. 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. Bacteria normally reproduce by binary fission. Once cell division begins. .24 Growth on MacConkey agar of strain 5 From the overall biochemical test result. each of these cells dividing so that four cell form. The curves consist of 4 phases which is the lag phase. Binary fission is a prokaryotic cell reproduction by division into two daughter cells. From Figure 4. and so forth in geometric progression. the stationary phase and the death phase.

It is an advanced method compare to the conventional method that use many steps that involve the use of organic solvent.25 Cell dry weight analysis plot of strain 2 and strain 5 4. From the value. RNase solution. isopropanol. . specific growth rate (µ) for strain 2 is 0. The Kit is an easy and reliable method to purify gram positive bacteria genomic DNA.47 From the ln x analysis against time for strain 2 and strain 5.5 4.1 16S rRNA Sequencing Genomic DNA extraction Chromosomal DNA of strain 2 and strain 3 were extracted using Promega WizardTM Genomic DNA Purification Kit. DNA Rehydration solution and protein precipitation solution. Figure 4. its shows that strain 5 can grow faster than strain 2.520 (Appendix 2) and specific growth rate for strain 5 is 0. Nucleic Lysis Solution.5. lysozyme. The kit consist of EDTA.576 (Appendix 2). ethanol.

Isopropanol and ethanol would precipitate and concentrate the DNA.4 bonds of peptidoglycan bond. The visibility of the band obtained shown that the genomic DNA was pure enough for sebsequent PCR amplification purpose.48 Lysozyme was used to weaken the cell wall by breaking the β-1. 1 2 3 4 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. Protein Precipitation solution was added to remove protein but leaves the high molecular weight genomic DNA in solution. 60 minutes) . 80 volts.26). DNA Rehydration Solution was added to rehydrate the DNA and was stored at -20oC to prevent from contamination and degradation of the DNA. 45 watts. First replicate of strain 2 and first replicate of strain 5 were selected for PCR amplification. The use of Nucleic Lysis solution is to weaken and lyse the nucleic acid.000 bp in size. The bands that present indicate that the DNA isolated were more than 10. Agarose gel electrophoresis was used to analyze the isolated genomic DNA to determine the success of the isolation (Figure 4. The addition of RNAse was to remove RNA that contaminating the genomic DNA extract.26 Agarose gel analysis of genomic DNA (1% w/v agarose. 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 annealing temperature (Ta) for the primer is 55oC.67 Concentration (µg/µl) 1.0. The ratio (A260/A280) less than 1.01167 0. 2003). Table 4.4.4 : Quantitative analysis of mixed culture genomic DNA Replicate Strain 2 (1) Strain 5 (1) A260 A280 A260/280 1.13 indicated with four .5.0 indicate that the genomic DNA is highly purify (Adams.00966 From Table 4.64 1.49 Purity and concentration of genomic DNA was determined spectrophotometrically by using a Varian Cary 100 model UV-Vis Spectrophotometer.343 0.4 summarizes the quantitative analysis of genomic DNA. ratio between A260 and A280 (A260/A280) was calculated. 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.7 is considered not pure enough and indicated to the presence of contamination such as protein and phenol.02686 0. 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. Table 4. Based on the genomic isolation.7 – 2.01640 0. This could be cause by contamination that presence in the genomic DNA.584 0. To calculate the purity of genomic DNA. Band on the agarose gel was shown in Figure 4.2 PCR amplication of 16s rRNA gene fragment Amplifications of the DNA samples were carried out using pA and pH primer. 4.86 11. The readings of 1.7-2.20 Dilution factor (DF) 103 103 A260 x DF 26. 1 µg/µL of template DNA from strain 2 and 3 µg/µL of template DNA was used to start of the PCR reaction. 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.

27 Agarose gel analysis of PCR products (1% w/v agarose. Figure 4.50 clear and strong bands with approximately 1500 bp in length were observed. 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. 1 2 3 4 DNA Marker: MassRulerTM DNA Ladder Mix. This indicated that the region of 16S rRNA was successfully amplified using pA and pH primers. . This maybe becaused by the lost during the purification step. 45 watts. 80 volts. 60 minutes) 4.14 show that the success of PCR products from as visualized under UV lamp.3 Purification and qualitative PCR product analysis Purification of the PCR reaction was done using the Wizard® SV Gel & PCR Clean-Up System (Promega).5. The band showed on the agarose gel appeared to be concentrated however replicate one of strain 2 did not show any visible band.

4. 45 watts.51 1 2 3 4 DNA Marker: MassRulerTM DNA Ladder Mix. The best scores gives similarity of 82% and this suggested that the bacteria might be closely related to genus Bacillus species. 60 minutes) 4.1 BLASTn analysis BLASTn performed pairwise comparison of DNA sequences and align to determine the homology of the query sequence.5. This result occurs because the PCR product that was sent is low in purity and also the concentration. However. 80 volts. strain 5 did not produce any result might due to the sequence result of the purified product. 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.5.4. The blasting result for strain 2 showed 100 bases quried. .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.

52 4.4.5.29 Phylogenetic tree processed and illustrated by Tree View . From Figure 4. Clostridium perfringens ABOO01 Strain 2 Bacillus thuringiensis EF63321 Pseudomonas sp EU557337 Bacillus sp EF633269 Bacillus cereus EF633204 Figure 4. the sequences were subjected to ClustalX program. To view the phylogenetic tree produce after running ClustalX analysis of the sequence. Phylogenetic tree is a graphical representation of the evolutionary relationship among a group of organisms or genes. Tree View was used.2 ClustalX After the BLASTn.29. tree showed that strain 2 might be related to Bacillus species that are known can be producing PHA. ClustalX program combines a good hierarchical method for multiple sequence alignment with an easy to use interface and for preparing phylogenetic tree.

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. strain 2 and strain 5 are from Bacillus species and from phylum acinetobacter respectively. bacterial strains 2 and 5 produce similar amount of PHA of 1.1 Conclusion The efficiency of PHA produce by bacteria depends on the species and how the nutrient is given to the bacteria. The biochemical tests are able to differentiate the two strains by several tests that have been done. Although other strains screened were able to produce PHA. . Different species of bacteria can be identified from the identification of the shape. Using minimal salt media with the supplementation of glucose as carbon source.CHAPTER 5 CONCLUSION AND FUTURE WORK 5. color and sizes of the colony produce.49 mg/L respectively. Molecular identification supports the result of strain 2. From the biochemical testing and gram staining results. 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.

4. . study can also conducted on the effectiveness of the degradability function of the PHA. a mixture of 16S rRNA fragments with different sequences will resolve into a distinct pattern of bands. The gene responsible for the production can be identified and further studied. 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. In addition. temperature and availability of oxygen. Using DGGE. Besides researching for production of PHA by bacteria. These factors can be manipulated to check whether the production can be enhanced and high productivity can be accumulated. 3. Using several other carbon source and environmental factor such as pH.2 Future Work There are several improvements and further study can be suggested: 1. Genetic engineering method can be introduced in order to higher the limits of production of PHA. PCR and DGGE studies have confirmed that standard culture techniques can be poor indicators of the composition of natural microbial populations.54 5. 2. 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.

Steinbuchel. A. Chavarie. M.-hydroxyvalerate copolymers. Berger. PHB recovery by hypochlorite digestion of non-PHB biomass. Calculation and Other Quantitative Skillsfor Use at the Bench Chapter 5. FEMS Microbiol.. Ramsay. G. (1993). Alexander Steinbiichel. F’EMS Microbiology Letters. Redwood City. metabolic role. In Lab Math: A Handbook of Measurement. 563. Diversity of bacterial polyhydroxyalkanoic acids. and industrial uses of bacterial polyhydroxyalkanoates. E. 127: 219-228 Adams.. Valentin (1995).. Tech. (1989). 54: 450-472. Byrom. (1992). Microbial ecology: fundamentals and applications. 1. The Benjamin and Cummings Publishing Company..S. 3: 227-232. E. A. Biotechnol. J. Occurrence. Dawes. Ramsay. NY : Cold Spring Harbor Laboratory Press. Biosci. D. A.55 REFERENCES A. (2003).. Braunegg. and Bartha. 127-145 Anderson. Rev. Atlas. R. Henry E. Microbiol. D. pp 1–24. C. . 103: 247-250. Lett. J. A. (2001). (1990). Perspectives for biotechnological production and utilization of biopolymers: metabolic engineering of polyhydroxyalkanoate biosynthesis pathways as a successful example. B. Macromol. R. metabolism. Production of poly.

P. Jean F. H. L.. Microbial degradation of synthetic polymers.Y.F. Chang. Slepecky (1960). Science Direct. G. Hahn. A. H. 3336. Chua.56 Byrom.. 5-33. Ramachandriah Shamala (2006). 26: 6565–6578. (1994).145 John H.. Kim. . (2005). D. Extraction of polyhydroxyalkanoate from Sinorhizobium meliloti cells using Microbispora sp. Patent 4. In: Frey et al. Biochemical test for identification of medical bacteria. Potential of biodegradable plastics as environmentally friendly substitute for conventional plastics in Hong Kong. Mac Faddin. USA.. Colorado. Ralph A.Q. P. 293-338..). Optimization of microbial poly (3-hydroxybutyrate) recovery using dispersions of sodium hypochlorite solution and chloroform. Ho. (1980). Chang. P. B. Separation process. Yu. Polyhydroxyalkanoates. (1994). The application of polyhydrxyalkanoates as tissue engineering materials. Biomaterials.. S. S. Bioeng. K.S. Culture and its enzymes. Lim. N. Assay of poly-β-hydroxybutyric acid. T. G. K. Chen.. Presented in the 17th Symposium on Biotechnology for Fuels and Chemicals.910. Cain RB (1992). Law.Wu.. Holmes. 1471-1475. (eds) Microbial Control of Pollution. Y. Plastics from microbes: microbial synthesis of polymers and polymer precursors. (1995b). 48th Symposium of the Society for general microbiology at University of Cardiff. May 1995. (1990). Biotechnol. Kshama Lakshman. U. In: D.H. Q. Xing. Hanser Munich. Second Edition. Illinois.. B. 44: 256-261.. S. Mobley (ed.

4-hydroxybutyrate: a strong flexible absorbable biomaterial.. Fratzke A.D. 16: 97–105. Nawrath. 49: 1-14. Bacterial Polyhydroxyalkanoates. Surampali (2006)... 43804387. NY... Microbiology: A Human Perspective (5th Edition). Starch esters as biodegradable plastics: Effects of ester group chain length and degree of substitution on anaerobic biodegradation.P. 57: 678-685. K. N. D. Bailey TB (1991). Y. Lee SY (1996).D. 17: 848852. (2003). Moens L. (1995). 255 O’Leary. 423-429. Williams. Production of polyhydroxyalkanoates. Kelley S (1995). in bacteria and plants. S. Yan. O’Connor. Genetic characterization of Accumulation of Polyhydroxyalkanoate from Styrene in Pseudomonas putida CA-3. S. Poirier. Appl. Roberts. Polymer production by bacterial strains isolated from activated sludge treating municipal wastewater. R.57 Lee B. Enzyme and Microbial Tech. 13(2): 142-150. Goff. A. Biochem.Y. Biotechnol. Environ. Rivard C. Roberts K. Pometto III AL. Bala Subramanian S. Dobson.F. Biodegradation of degradable plastic polyethylene by Phanerochaete and Streptomyces species. J. Medical applications of poly. (2005). C. P. Microbiol. Martha Nester (2007).D. Eng.E. R. a family of biodegradable plastics and eleastomers... Journal of Biotechnology. Martin. Nester.W. Brigham J.. Applied and Environmental Microbiology. Ward. . McGraw-Hill Education. C. Tyagi. Bioeng. and Somerville. Anderson. M.

J. Salehizadeh. Sang Yup Lee. Abe. structure and properties of polyhydroxyalkanoates: biological polyesters. Lafferty. H. Doi. pp 283-294.. Int. Synthesis. Macromol. 124-213 Sudesh. Stockton. (2000). Y.. Sci. Macromol. New York. M. K.. (1994). Plastic bacteria? Progress and prospects for polyhydroxyalkanoate production in bacteria. Y. Polym. Steinbuchel. Polyhydroxyalkanoic acids. 107-113. In: D. Biochemical Engineering Journal.. I. 31-36. Byrom (ed. 431-438. G.. 22: 261-279 Sang Yup Lee (1996). Biol. Tomoyasu Kichise. M. J..58 Saito. Yoshiharu Doi (2002). (2004). Mikrobiol. Gauss. H. R. The isolation of mutants not accumulating poly-P-hydroxybutyric acid. Doi. C. Schlegel. Jong-il Choi. Production of polyhydroxyalkanoates by mixed culture: recent trends and biotechnology importance. Production of polyhydroxyalkanoate (PHA) from renewable carbon sources in recombinant Ralstonia eutropha using mutants of original PHA synthase. Arch. Recent advances in polyhydroxyalkanoate production by bacterial feremntation: mini review. Heng Ho Wong (1998). . 1970. Biotechnology Advances. (1991). 71. 16: 99–104. Prog. Hirofumi Nakamura. and Van Loosdrecht. Int. A. Microbial synthesis and properties of poly(3hydroxybutyrate-co-4-hydroxybutyrate) in Comamonas acidovorans. Biol.). Y. 25: 1503-1555. Biomaterials: novel materials from biological sources. Elsevier Science Ltd. H. Seiichi Taguchi.

FEMS Microbiol. using the polymerase chain reaction (PCR). Sherwood. Letter. 271 Yamane.Kshama (2002). Guo-Qiang Chen. S. 103: 257-264. Woolverston (2008). Identification of polyhydroxyalkanoate (PHA)-producing Bacillus spp. Zengming Zhang (1998). L. Cultivation engineering of microbial bioplastics production. McGraw-Hill Education.V. Guang Zhang. Rev. Production of novel Polyhydroxyalkanoates by Pseudomas stutzeri 1317 from glucose and soybean oil. Vijayendra. 45-49.N.R. T. Journal of Applied Microb. Wennan He. Harley and Kleins’s Microbiology (7th edition). . Willey. Weidong Tian. 369-374. 1992. Chandrashekar. Shamala. FEMS Microb. A.59 T. Prescott.

100 Strain 5 0.048 0.60 2.10 2.122 0.93 1.87 73.036 0.365 0.250 0.97 4.0 1.29 4.0 3.46 2.5 6.5 9.5 7.48 0.65 13.511 1.816 1.61 13.11 17.03 2.150 8.761 1.5 5.74 0.02 -0.28 0.018 0.30 8.27 0.88 4.20 0.31 -0.71 -1.0 4.81 0.770 8.07 0.78 5.980 7.21 7.209 0.43 15.43 3.949 5.031 0.0 6.305 1.24 2.43 4.5 4.24 4.82 -1.5 3.30 3.65 1.20 59.00 Strain 5 2.17 -1.044 0.27 -1.30 81.78 2.50 87.09 1.0 7.72 2.178 0.521 0.83 1.50 2.57 2.60 ln X Strain 2 -1.787 7.0 8.60 APPENDICES APPENDIX A Table : OD at 600nm analysis.44 .40 4.58 0.31 0.44 0.05 19.60 81. cell dry weight and ln X value of strain 2 and strain 5 Time Interval (h) 0.50 81.03 4.400 8.224 0.36 0.5 2.49 56.460 X (mg/mL) Strain 2 0.230 0.310 6.0 OD (600nm) Strain 2 0.5 8.92 0.0 2.028 0.47 4.73 -0.22 2.99 2.620 5.378 0.89 1.26 4.10 69.33 1.960 8.29 2.5 1.80 70.050 8.09 4.39 4.730 0.343 1.0 0.027 0.093 0.130 8.165 1.18 0.00 84.70 84.243 0.65 7.16 11.0 5.39 Strain 5 0.

35 4.90 69.340 81.30 81.46 4.970 8.600 8.40 4.690 7.45 4.30 4.44 4.44 4.24 4.39 4.61 9.60 81.5 8.40 4.440 8.5 12.160 8.100 7.430 8.43 4.0 10.10 76.5 10.150 8.70 86.33 4.31 .780 7.10 84.0 11.0 12.00 86.450 8.00 77.50 81.80 74.40 84.610 8.5 11.90 75.50 84.5 13 13.34 4.40 4.110 7.590 6.

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 .

2848 0.3879 1.6934 1.1496 10.3235 4.4935 0.1087 6.2054 0.97910 4.4503 0.36460 6.2816 0.6603 0.6079 .7455 23.63 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 10.4909 0.3163 0.18360 9.41500 3.90250 4.01330 23.42380 Amount of PHA (mg/L) 0.

5% solution of dimethyl-α-naphthylamine in 5N acetic acid *Possible and can be potentially carcinogen. Reagent A: 0. Reagent B: 0. . 3.0 1.8% solution of sulfanic acid in 5N acetic acid. Beef extract peptone broth: Composition Beef extract Peptone Potassium nitrate Amount (g/L) 3.0 5.64 APPENDIX D 1.0 2.

5 mL of the medium was dispensed in sterile test tubes. 5 mL of filter sterilize glucose was added aseptically from the stock solution of glucose. pH was adjusted to 7. 3 mL of bromothymol blue was added to the medium.0 5. . All the chemical compositions were mixed in distilled water and stirred it to dissolve it. 3. 50 mL of the medium was autoclaved at 121oC for 15 minutes.5 Method: 1. 5. Later. 4. 2.0 0.3 2.65 APPENDIX E Preparation of OF-Glucose Medium Composition Peptone (pancreatic digest of casein) Sodium chloride di-Potassium hydrogen phosphate anhydrous Agar Bromothymol blue (1.0.5% w/v stock) Glucose (10% w/v stock) Amount (g/L) 2.

0 1. FeSO4 Sodium thiosulphate.024 Method: 1.0 0.2 5. NaCl Agar Phenol red Amount (g/L) 3. All the chemical compositions were dissolved into 100 mL of distilled water and pH was adjusted to 7.0 20.66 APPENDIX F Preparation of Triple Sugar Iron (TSI) Agar Composition Beef extract Yeast extract Peptone Lactose Sucrose Glucose Ferrous sulphate. 2. Na2S2O3 Sodium chloride.0 12.2 0.4.0 0.0 3. All the tubes were then autoclaved at 121oC for 15 minutes and cooled to room temperature in slanted position. The medium was then poured into test tubes each containing approximately 5 mL to make long slant agar. 3. .0 10.0 10.

All the tubes were then autoclaved at 121oC for 15 minutes and cooled to room temperature in slanted position.0 2.0 10. The medium was then poured into test tubes each containing approximately 5 mL to make long slant agar. 3. All the chemical composition was dissolved into 1000 mL of distilled water and the pH was adjusted to 7.4.0 Method: 1.5% w/v) Agar Amount (g/L) 1. . 2.0 1.0 0.2 5.0 20.67 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.

2. the solution was filtered and stored in brown bottle Preparation of Kovac’s Reagent Method: 1. A 10 g of p-dimethylaminoensaldehyde dissolved in 150 mL of pure isoamyl alcohol. Finally. 2. . Then. 3.0 g of potassium iodide (KI) was dissolved in 100 mL distilled water. A concentrated HCl is then slowly added into the aldehyde-alcohol mixture. 1.0 g of crystal iodine (I2) was added slowly and shake it until dissolved in the mixture. 5.68 APPENDIX H Preparation of Lugol’s Iodine Method: 1.

The medium were then poured into test tubes each containing approximately 5 mL and solidify it in slanted position.0 5.0 0.69 APPENDIX I Preparation of Christensen urea agar slant Composition Monopotassium Sodium chloride Peptone Glucose Urea Phenol red Agar Amount (g/L) 2.0 1. 2. All the compositions were dissolved in 900 mL of distilled water except urea and autoclaved it at 121oC for 15 minutes.01 15 Method: 1. 20 g urea and 1 g glucose were rehydrated in 100 mL distilled water and filter sterilized it using syringe.0 1. The urea and glucose solution that have been filtered was added aseptically into the sterile medium. 4. 3. .0 20.

0 5. 2.8.0 1. The medium was then autoclaved at 121oC for 15 minutes . All the chemical composition above was dissolved in 1000 mL of distilled water and the pH was adjusted to 6.70 APPENDIX J Preparation of Tryptone Broth medium Composition Tryptophan Yeast extract Sodium chloride di-Sodium hydrogen phosphate Amount (g/L) 2. The medium were then poured into test tubes each containing approximately 4 mL. 3.0 Method: 1.0 3.

0 1% (v/v) Method: 1.15 20. All the chemical composition above was dissolved in 1000 mL of distilled water and the pH was adjusted to 6. 2.8.0 0. The medium was then autoclaved at 121oC for 15 minutes .0 10.71 APPENDIX K Preparation for lipase activity Composition Yeast extract Bacto-Tryptone Methylene blue Agar Tween 80 Amount (g/L) 5.