screening and characterization of pha producing bacteria from activated sludge

SCREENING AND CHARACTERIZATION OF PHA-

PRODUCING 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 : 29 APRIL 1986

Title : SCREENING AND CHARACTERIZATION OF PHA-PRODUCING

BACTERIA FROM ACTIVATED SLUDGE

Academic Session : 2007/2008-2

I declare that this thesis is classified as :

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 DR. ADIBAH YAHYA

(NEW IC NO. /PASSPORT NO.) NAME OF SUPERVISOR

Date : APRIL 2008 Date : APRIL 2008

CONFIDENTIAL (Contains confidential information under the

Official Secret Act 1972)*

RESTRICTED (Contains restricted information as specified by

the organization where research was done)*

OPEN ACCESS I agree that my thesis to be published as online

open access (full text)

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

ii

“I hereby declared that I have read this thesis and in my

opinion this thesis is sufficient in terms of scope and quality for the

award of the degree of Bachelor of Science (Pure Biology).”

Signature : ..………………………….

Name of Supervisor : Dr. Adibah Yahya

Date : 16th April 2008

iii

I hereby declare that this thesis entitled ‘Screening and Characterization of PHA-

Producing Bacteria from Activated Sludge’ is the result of my own research except as in

cited references. The thesis has not been accepted for any degree and is not concurrently

submitted in candidature of any other degree

Signature : ………………………………..

Name : Himi Husme Bin Abd Majid

Date : 16th April 2008

iv

In loving memory of my dad, Abd. Majid Bin Mohd. Salleh

For my beloved and supporting mom, Normah Mamat, my dearest sister,

Nadiratul Noziana and my step dad, Azmi bin Abdul Wahab.

…….Thank you very much……

v

ACKNOWLEDGEMENT

Firstly, I would to give my gratitude to my project supervisor, Dr. Adibah Yahya,

for her commitment, time spend, encouragement, support and guidance in completion of

this project throughout the year. It would be difficult for me to complete this project

without her help and advice.

Next, I would also like to thank Assc. Prof. Dr. Zaharah Ibrahim for her advice

and ideas and also the caring attitude all throughout the year while completing this

project. A lot of thank to lab assistants, Puan Fatimah, Puan Radiah, Encik Awang and

Encik Yus for their cooperation in providing all the lab instruments and guidance while

using the instruments. Not to forget Cik Fareh Nunizawati that has given me her advice

and support to do the experiments efficiently.

I would also like to express my appreciation and thanks to my loving and caring

parents, my lovely sister and all the people and friends that help me complete this

project.

Thank you very much………………

vi

ABSTRACT

Eleven strains of bacteria previously isolated from activated sludge in PHA-producing

reactor were used in this study. The activated sludge contains mix culture of bacteria

that were able to use various types of organic compound presence in the sludge for

growth and PHA (polyhydroxyalkanoate) production. In this study, the bacteria were

screened for their ability to produce PHA in a minimal medium supplemented with

glucose as carbon source and grow at 30oC. Results indicated that all bacteria tested

were able to produce PHA, though only four strains showed enhanced PHA production.

However, only two strains coded strain 2 and strain 5 were selected for further

identification due to higher and almost similar concentration of PHA produced which is

1.4909 mg/L and 1.4935 mg/L respectively. Morphology characteristic of strain 2 and

strain 5 showed that both were clearly distinguish by their shape of colonies. Strain 2

was observed and showed white flat colony with clear edge zone at each of the colony.

In contrast, the colony of strain 5 appeared to be more like fungi and was highly slimy.

Cellular characterization showed that strain 2 was a rod shape cell and strain 5 was

coccobacilli cell. From the biochemical test, strain 2 may belonged to Bacillus species

and strain 5 can be a Acinetobacter species. The 16s rRNA characterization of the

bacteria showed that strain 2 also belonged to Bacillus species (82%).

vii

ABSTRAK

Sebelas jenis bakteria yang sebelumnya dipencilkan daripada selut beraktivasi di

dalam reaktor yang menghasilkan PHA telah digunakan dalam kajian ini. Selut

beraktivasi tersebut mengandungi campuran kultur bacteria yang berkebolehan untuk

menggunakan pelbagai jenis bahan organik yang terdapat di dalam selut untuk

pertumbuhan dan penghasilan PHA (Polyhydroxyalkanoate). Dalam kajian ini, bakteria

tersebut dikesan kebolehannya untuk menghasilkan PHA dalam medium minimum yang

ditambah dengan glukosa sebagai sumber karbon dan tumbuh pada 30oC. Keputusan

menunjukkan semua bakteria yang diuji berkebolehan untuk menghasilkan PHA.

Walaupun begitu, hanya empat jenis bakteria mununjukkan penghasilan PHA yang

tinggi. Walaubagaimanapun, hanya dua jenis bakteria berkod strain 2 dan strain 5 telah

dipilih untuk pengenalpastian lanjutan merujuk kepada penghasilan PHA yang lebih

tinggi dan kepekatan yang hampir sama iaitu 1.4909 mg/L dan 1.4935 mg/L setiapnya.

Pencirian morfologi bagi strain 2 dan strain 5 menunjukkan kedua-duanya dapat

dibezakan degan jelas berdasarkan bentuk koloni mereka, Strain 2 telah diperhatikan dan

menunjukkan koloni putih rata dengan zon lutsinar pada setiap koloni. Sebaliknya,

koloni bagi strain 5 adalah menyerupai fungi dan sangat berlendir. Pencirian pada

peringkat sel menunjukkan strain 2 berbentuk rod dan strain 5 berbentuk rod dan bulat.

Daripada keputusan biokimia, strain 2 dikenalpasti tergolong dalam spesis Bacillus dan

strain 5 dari spesis Acinetobacter. Daripada pencirian menggunakan teknik 16S rRNA,

strain 2 dikenalpasti tergolong dalam sepsis Bacillus (82%).

viii

CONTENTS

CHAPTER TITLE PAGE Title i

Supervisor’s Declaration ii

Declaration iii

Dedication iv

Acknowledgements v

Abstract vi

Abstrak vii

Contents viii

List of Tables xii

List of Figures xiii

List of Abbreviation xv

List of Appendices xvii

1 INTRODUCTION 1

2 LITERATURE REVIEW 3

2.1 Polyhydroxyalkanoates (PHA) 3

2.2 Chemistry of the PHAs 4

2.3 Physical Properties of PHAs 5

2.4 The biology of PHA 7

2.5 Biosynthesis of PHA 7

2.7 Recovery of PHA 10

2.8 Carbon Substrate and Yield 10

ix

2.9 Other Types of PHA and Application 12

2.10 Characterization and Identification 13

2.10.1 16S rRNA 13

2.10.2 Polymerase Chain Reaction 13

2.10.3 Agarose Gel Electrophoresis 14

2.10.4 Gram Staining 15

2.10.5 Biochemical Characterization 16

3 MATERIALS AND METHODS 18

3.1 Experimental Design 18

3.2 Microorganisms 19

3.2.1 Morphology Characterization of Bacteria 19

3.2.2 Preparation of Inoculums 20

3.2.3 Preparation of Stock Culture 20

3.3 Media preparation 20

3.3.1 Nutrient Broth 21

3.3.2 Nutrient Agar 21

3.3.3 Mineral Salt Medium 21

3.4 Screening of PHA Producer 22

3.5 Characterization and Identification of

Potential PHA Producing Bacteria 23

3.5.1 Biochemical Characterization 23

3.5.1.1 Catalase Test 23

3.5.1.2 Cytochrome oxidase 23

3.5.1.3 Nitrate reduction 24

3.5.1.4 Citrate Test 24

3.5.1.5 Triple Sugar Iron Test 25

3.5.1.6 Starch hydrolysis 25

3.5.1.7 OF-Glucose Test 25

3.5.1.8 Gelatin liquefaction Test 26

3.5.1.9 Urease Test 26

x

3.5.1.10 Indole Test 26

3.5.1.11 Motality 27

3.5.1.13 Lipase Test 27

3.5.1.14 Voges Proskauer Test 27

3.5.2 Molecular Characterization 28

3.5.2.1 DNA Extraction 28

3.5.2.2 PCR Amplification of 16S rRNA

Fragment 29

3.5.2.3 Purification of the Amplified 16S

rRNA Fragment 30

3.5.2.4 Agarose Gel Electrophoresis 31

3.5.2.5 Sequencing of the Amplified 16S

rRNA Fragment 31

3.5.2.6 Phylogenetic Tree Construction 31

3.6 Analytical Methods 32

3.6.1 Determination of PHA 32

3.6.2 Determination of Bacterial Growth 32

4 RESULT AND DISCUSSION 33

4.1 Screening of Potential PHA Producer 33

4.2 Colony and Cellular Morphologies Characterization 35

4.2.1 Gram Staining 40

4.3 Biochemical Characterization 41

4.4 Bacterial growth analysis 46

4.5 PCR Amplication Method for Microbial

Identification 47

4.5.1 Genomic DNA Extraction 47

4.5.2 PCR Amplication of 16s rRNA Gene Fragment 49

4.5.3 Purification and Qualitative PCR Product

analysis 50

4.5.4 DNA Sequence analysis 51

xi

4.5.4.1 Blastn Analysis 51

4.5.4.2 ClustalX 52

5 CONCLUSION AND FUTURE WORK 54

5.1 Conclusion 54

5.2 Future Work 54

REFERENCES 55

APPENDICES 60-71

xii

LIST OF TABLES

TABLE NO. TITLE PAGE

2.1 Classification of microbial PHAs according to 6

different criteria

2.2 Effect of substrate cost and P(3HB) yield of the

production cost of P(3HB) 11

2.3 Possible application of PHA 12

3.1 Sequences of eubacterial 16S rDNA universal primers 29

3.2 PCR reaction mixtures 30

3.3 Gradient PCR cycle profile 30

4.1 Colony morphologies of pure colony on Agar plate

culture. 37

4.2 Cellular characterization of strain 2 and strain 5 40

4.3 Summary of Biochemical test result 41

4.4 Quantitative analysis of mixed culture genomic DNA 49

xiii

LIST OF FIGURES

FIGURE NO. TITLE PAGE

2.1 General structural formula of PHA 5

2.2 Principle of biosynthesis of bacteria in bacteria. Three

relevant phases of biosynthesis of PHA are shown 9

3.1 Schematic representation of overall experimental setup 18

4.1 Amount of PHA produce by bacterial strains 34

4.2 Colony morphology of Strain 1 38











4.13 Cellular morphology of strain 2 40

4.14 Cellular morphology of strain 5 40

4.15 Nitrate reduction test of Strain 2 42

4.16 Nitrate reduction test of Strain 5 42

4.17 Starch hydrolysis test on Strain 2 43

4.18 Starch hydrolysis test on Strain 5 43

4.19 Negative result of urease test of strain 2 44

xiv

4.20 Positive result of urease test of strain 5 44

4.21 Gel liquefaction test of strain 2 45

4.22 Gel liquefaction test of strain 5 45

4.23 No growth on MacConkey agar of strain 2 46

4.24 Growth on MacConkey agar of strain 5 46

4.25 Cell dry weight analysis plot of strain 2 and strain 5 47

4.26 Agarose gel analysis of genomic DNA (1% w/v

agarose, 80 volts, 45 watts, 60 minutes) 48

4.27 Agarose gel analysis of PCR products (1% w/v

agarose, 80 volts, 45 watts, 60 minutes) 50

4.28 Agarose gel electrophoresis of the purified PCR

products (1% w/v agarose, 80 volts, 45 watts, 60 minutes) 51

4.29 Phylogenetic tree processed and illustrated by Tree View 52

xv

LIST OF ABBREVIATIONS

A – Absorbance

BLAST – Basic Local Alignment Search Tool

bp – base pair oC – degree Celcius

dH2O – distilled water

dsDNA – double stranded DNA

DNA – Deoxyribonucleic acid

dNTP – deoxynucleotide triphosphate

EDTA – Ethylenediaminetetraacetic acid

EPS – Extracellular polysaccharide

H2SO4 – Sulphuric acid

HCl – Hydrogen chloride

g/L – gram per litre

kbp – kilobase pairs

µL – microlitre

min – minutes

h – hours

mL – mililitre

M – Molar

ng/µL – nanogram per microlitre

nm – nanometer

NCBI – National Center of Biotechnology Information

NaOH – sodium hydroxide

OH – Hydroxyl

% - percent

xvi

PCR – Polymerase Chain Reaction

PHA - Polyhydroxyalkanoate

rRNA – ribosomal Ribonucleic Acid

sp. – species

Ta – Annealing temperature

Tm – Melting temperature

UV - Ultra violet

V – Volts

w/v – weight per volume

v/v – 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

B OD 600nm analysis plot of strain 2 and strain 5 62

B ln X analysis plot of strain 2 62

B ln X analysis plot of strain 5 62

C Value of OD and amount of PHA produce by

bacterial strains 63

D Beef extract peptone broth 64

E Preparation of OF-Glucose Medium 65

F Preparation of Triple Sugar Iron (TSI) Agar 66

G Preparation of Simmons Citrate Agar 67

H Preparation of Lugol’s Iodine 68

H Preparation of Kovac’s reagent 68

I Preparation of Christensen urea agar slant 69

J Preparation of Tryptone Broth medium 70

K Preparation for lipase activity 71

CHAPTER 1

INTRODUCTION

In response to increasing public concern about the harmful effects of

petrochemical derived plastic materials in the environment, many countries are

conducting various solid-waste management programs, including plastic waste reduction

by developing biodegradable plastic materials. These biodegradable plastic materials

must retain the desired material properties of conventional synthetic plastics and should

be completely degraded without leaving any undesirable residues when discarded.

Polyhydroxyalkanoates, (PHAs) are polyesters of various hydroxyalkanoates

which are synthesized by numerous microorganisms as an energy reserve material,

usually when an essential nutrient such as nitrogen or phosphorus is limited in the

presence of excess carbon source. PHAs are considered to be strong candidates for

biodegradable polymer material because they possess material properties similar to

various synthetic thermoplastics and elastomers currently in use (from polypropylene to

synthetic rubber) and upon disposal, they are completely degraded to water and carbon

dioxide (and methane under anaerobic conditions) by microorganisms in various

environments such as soil, sea and lake water.

This study was purpose for finding the best bacteria that grow and produce high

concentration of polyhydroxyalkanoate, (PHA). PHA are same polymer as plastics but

it can it degrade more and effectively than normal plastics that are made from petroleum.

Although many bacteria can produce PHA when supplied with the suitable growth

condition and carbon substrate, not all the bacteria can produce high production of PHA.

2

The objectives of this study are:

1. To screen the potential PHA-producing bacteria.

2. To characterize selected potential bacteria using biochemical and molecular

method

3

CHAPTER 2

LITERATURE REVIEW

2.1 Polyhydroxyalkanoates (PHA)

Polyhydroxyalkanoates (PHAs) are the polymers of hydroxyalkanoates that

accumulate as carbon/energy or reducing-power storage material in various

microorganisms (Salehizadeh and Van Loosdrecht, 2004). PHAs are stored in the

bacterial cytoplasm as inclusion bodies (Lee, 1996) and they are synthesized and

accumulated intracellularly as distinct granules, usually under unfavorable growth

conditions, such as feast and famine regime, limitation of nitrogen, phosphorus, sulphur,

magnesium or oxygen in the presence of excess carbon source (Poirier, 1995).

Basically, PHAs can be broadly subdivided into three groups based on the

number of carbon atoms present in its monomer units (Steinbuchel, 2001),:

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

).

(b) Medium-chain-length PHAs consisting of 6-14 carbon atoms (PHAMCL

).

(c) Long-chain-length PHAs consisting of more than 14 carbon atoms

(PHALCL

).

4

2.2 Chemistry of the PHAs

Of all the biodegradable plastics being studied, those that have generated the

most interest are the poly(3-hydroxyalkanoates) or PHAs which are made by bacteria.

Like all plastics, PHAs are polymers, long molecules made up of many small subunits

(monomers) which have been joined together. These water-insoluble storage polymers

are biodegradable, exhibit thermoplastic properties and can be produced from renewable

carbon sources. The composition of the polymer synthesized is governed by two main

factors, i.e. the bacterial strain being used and the carbon source utilized to grow the

bacteria. (S.P. Valappil, 2007)

In the case of PHAs, the monomers are 3-hydroxyalkanoates. An alkanoate is

simply a fatty acid which is a linear molecule containing just carbon and hydrogen (an

alkane) with a carboxyl group at one end (making an alkanoate). Furthermore, these

monomers have a hydroxyl group (OH) at the 3rd carbon (what used to be called the beta

position), making these beta or 3-hydroxyalkanoates. The hydroxyl group of one

monomer is attached to the carboxyl group of another by an ester bond; these plastics are

thus polyesters.

As is shown in Figure 2.1, the polyester linkage creates a molecule which has 3-

carbon segments separated by oxygen atoms. The remainder of the monomer becomes a

sidechain off the main backbone of the polymer. Most of the PHAs encountered in

nature are poly(beta-hydroxybutyrate) (PHB), in which the monomer unit is

hydroxybutyric acid and the side chain is a methyl group. Other monomer units occur in

nature, and many others can be produced in the laboratory by feeding unusual carbon

sources to bacteria. Most PHAs, even what we call PHB, are actually copolymers, and

contain some amount of another type of monomer unit.

5

Figure 2.1 General structural formula of polyhydroxyalkanoate (PHA)

2.3 Physical Properties of PHAs

The composition of the PHA has a direct effect on the physical properties of the

plastic, in. PHB, with its short methyl side chain, is a very crystalline and very brittle

polymer. Industrially, it is difficult to use because the temperature at which it melts is

very close to the temperature at which it begins to decompose. Its high degree of

crystallinity causes it to crack easily. As a result, the PHA used commercially is PHBV,

a copolymer of hydroxybutyrate and hydroxyvalerate (5 carbons long). PHBV is a

random copolymer, meaning that the monomer units do not occur in the chain in any

particular order. PHBV can still crystallize, but it produces a much more supple plastic

and melts at a lower temperature, making processing easier. PHB and PHBV have

properties similar to polypropylene, and bottles made from these polyesters feel just like

"normal" plastic. The flexibility increases with sidechain length throughout the PHA

family, largely because of a loss of crystallinity. Polymers composed mostly of

hydroxyoctanoate, an 8- carbon monomer, are elastic. Longer side chain polymers are so

soft that they are gummy or glue-like. This remains one of the potential values of PHAs,

that by feeding bacteria an appropriate substance, a PHA with specific desirable

properties can be produced.

6

PHAs can be classified into various groups according to different criteria (Table

2.1). Among them, classification according to the monomer size, which refers to the

number of carbon atoms in the HA monomer, and the type of polymer is the most

common and will be described in detail here.

Table 2.1 : Classification of microbial PHAs according to different criteria (Luengo,

2003)

Biosynthetic origin

• Natural PHAs: produced naturally by microorganisms

from general substrates. i.e Poly(3-hydroxybutyrate)

P(3HB).

• Semisynthetic PHAs: requires the addition of unusual

precursors such as 3-mercaptopropionic acid to

promote the biosynthesis of poly(3-hydroxybutyrate-

co-3-mercaptopropionic) [P(3HB-co-3MP)].

Monomer size (depending on the number of carbon atoms in an HA monomer

• Short chain-length PHAs (SCL-PHA): contains 3-5

carbon atoms.

• Medium chain-length PHAs (MCL-PHA): contains 6-

14 carbon atoms.

Number of different monomers in PHAs

• Homopolymer: The polymerization begins with the

linkage of a small molecule or monomer through

ester bonds to thecarboxylic group of the next

monomer. A homopolymer is produced when single

monomeric units are linked together. i.e P(3HB).

• Heteropolymer: When two or more different

monomeric units are linked together, a copolymer is

formed. i.e P(3HB-co-4HB).

Chemical nature of the monomers

• PHAs containing aliphatic fatty acids. i.e P(3HB).

• PHAs containing aromatic fatty acids.

• PHAs containing both aliphatic and aromatic fatty

acids. i.e P(3HB-co-3MP).

7

2.4 The Biology of PHA

In nature, prokaryotic microorganisms respond to sudden increases in essential

nutrients in their usually hostile environment by storing important nutrients for survival

during prolonged period of starvation (Sudesh, 2000). PHAs are one such storage

compound. PHAs are usually produced when carbon sources are in excess. The carbon

sources are assimilated, converted into hydroxyalkanoate (HA) compounds and finally

polymerized into high molecular weight PHAs and stored as water insoluble granules in

the cell cytoplasm. PHAs are an excellent storage compound because their presence in

the cytoplasm, even in large quantities does not disturb the osmotic pressure of the cell.

PHA granules can be observed as refractile granules under phase contrast light

microscope. When thin sections of cells containing PHAs are viewed under transmission

electron microscope, the granules appear as electron transparent, discrete, spherical

particles with clear boundaries. The number and sizes of granules per cell differ

depending on the PHA-producer microorganisms and their growth stage. In Wautersia

eutropha (formerly known as Alcaligenes eutrophus), 8-13 granules per cell with sizes

ranging from 0.2-0.5 m were detected (Byrom, 1994).

PHA granules could be stained with Sudan Black (Schlegel, 1970) and more

specifically by Nile Blue A, exhibiting a strong orange fluorescence. Nile Blue A is a

more specific dye than Sudan Black B as it does not stain glycogen and polyphosphate.

Both stains however can stain lipid bodies.

2.5 Biosynthesis of PHA

Polyhydroxyalkanoates are polyesters of hydroxyalkanoates (HAs) having the

general structural formula shown in Figure 2.1. Numerous bacteria can synthesize and

accumulate PHAs as carbon and energy storage materials or as a sink for redundant

8

reducing power under the condition of limiting nutrients in the presence of excess

carbon (Steinbuchel, 1991).

Three metabolic phases of the biosynthesis of PHA in bacteria can be

distinguished (Figure 1.3). First, a carbon source suitable for biosynthesis of PHA must

enter the cell from the environment. This is achieved either by a specific transport

system located in the cytoplasmic membrane or by diffusion of the compound into the

cell. Second, anabolic or catabolic reactions, convert the compound into a hydroxyacyl

coenzyme A thioester which is a substrate of the PHA synthase. Third, PHA synthase,

which is the key enzyme of PHA biosynthesis, uses these thioesters as substrates and

catalyzes the formation of the ester bond with the concomitant release of coenzyme A.

At present it cannot generally be excluded that the PHA synthases also use other

thioesters of HA as substrates. Phase II is of most importance, since during this phase

the carbon source is converted into a suitable substrate for the PHA synthase. Many

bacteria are able to convert acetyl-CoA in two steps via acetoacetyl-CoA to D(-)-3-

hydroxybutyryl-CoA giving rise to poly(3HB). Regarding the application of precursor

substrates, the most simple type of reaction is the conversion of a HA, which is provided

as a carbon source to the cells, by a thiokinase or a coenzyme A transferase into the

corresponding HA-coenzyme A thioester, such as, for example, the conversion of 4HB

into 4-hydroxybutyryl-coenzyme A. If the carbon source is not a precursor substrate, and

if the carbon source is first converted into a central intermediate of metabolism, a

complex sequence of reactions may be required to obtain PHA consisting of HA other

than 3HB (Steinbiichel. 1995).

9

Figure 2.2: Principle of biosynthesis of bacteria in bacteria. Three relevant phases of

biosynthesis of PHA are shown

2.6 Production of PHA

Figure 2.2 Principle of biosynthesis of bacteria in bacteria. Three relevant phases of

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

In most bacteria PHAs are synthesized and intracellularly accumulated under

unfavorable growth conditions such as limitation of nitrogen, phosphorus, magnesium,

or oxygen in the presence of excess carbon (Anderson, 1990). It is, therefore, important

to develop cultivation strategies that can simulate these conditions for the efficient

production of PHA.

Some bacteria such as Alcaligenes latus and a mutant strain of Azotobacter

vinelandii are known to accumulate PHA during growth in the absence of nutrient

limitation. Selection of a microorganism for the industrial production of PHA should be

based on several factors including the cell’s ability to utilize an inexpensive carbon

source, growth rate, polymer synthesis rate, and the maximum extent of polymer

accumulation. The yield of PHA on carbon source is important not to waste substrate to

non- PHA material. An equation that predicts the overall yield of PHA on several carbon

sources has been derived and can be used for the preliminary calculation of PHA yields

(Yamane, 1992). Recovery of PHA should also be considered because it significantly

affects the overall economics.

10

2.7 Recovery of PHA

Following the fermentation, cells containing PHAs are separated by conventional

procedures such as centrifugation, filtration, or vortexing. After the biomass harvesting,

cells are disrupted to recover the polymers. A number of different methods have been

developed for the recovery of PHA. The first method that has most often been used

involves extraction of P(3HB) from biomass with solvent. The solvents employed

include chloroform, methylene chloride, propylene carbonate, and dichloroethane. Due

to the high viscosity of even dilute PHA solutions, about 20 portion of solvent are

required to extract 1 portion of polymer (Byrom, 1994). The large amount of solvent

required makes this method economically unattractive, even after the recycling of the

solvent (Holmes, 1994).

Several other methods that have been developed involve the use of sodium

hypochlorite for the differential digestion of non-PHA cellular materials (Berger, 1989).

Although this method is effective in the digestion of non-PHA cellular materials, it

causes severe degradation of P(3HB) resulting in a 50% reduction in the molecular

weight. The use of sodium hypochlorite together with chloroform significantly reduced

degradation of PHA (Hahn, 1994). It was suggested that chloroform immediately

dissolves the isolated P(3HB) by hypochlorite, and thus protects polymer from

degradation. Normally, polymer purity of greater than 95% is obtained by hypochlorite

treatment.

2.8 Carbon substrate and yield

Excluding the recovery process, the economics of PHA production are largely

determined by the substrate cost and PHA yield. The efficiency of substrate conversion

is important, and can be predicted from the physiology and biochemistry involved in the

PHA synthesis. Among the various nutrients in the fermentation medium, the carbon

source contributes most significantly to the overall substrate cost in PHA production. A

11

number of carbon sources, including carbohydrates, oils, alcohols, acids and

hydrocarbons, can be used by various bacteria (Yamane, 1993).

The theoretical yield (the yield based on the reaction stoichiometry) of P(3HB)

has been estimated for several of these carbon substrates (Yamane, 1993). Regeneration

of nicotinamide nucleotides, which are used as cofactor for PHA synthesis, has been

taken into account in this analysis. It was also suggested that the overall yield, which is

the yield in actual fermentation, would be roughly proportional to the theoretical yield

and PHA content.

Table 2.2 summarizes the cost of carbon substrate based on the theoretical yield.

Because of their low price, crude carbon substrates such as the cane and beet molasses,

cheese whey, plant oils and hydrolysates of starch (corn and tapioca), cellulose and

hemicellulose can be excellent substrates to several bacteria utilizing them.

Table 2.2 : Effect of substrate cost and P(3HB) yield of the production cost of P(3HB)

Substrate Approximate price (US$/kg) P(3HB) yield Substrate cost

Glucose 0.493 0.38 1.30

Sucrose 0.290 0.40 0.72

Methanol 0.180 0.43 0.42

Acetic acid 0.595 0.38 1.56

Ethanol 0.502 0.50 1.00

Cane Molasses 0.220 0.42 0.52

Cheese Whey 0.071 0.33 0.22

12

2.9 Other types of PHA and application

Beside PHA, there are many types of PHA. The material properties and hence

the application of the PHAs vary depending on the monomers composition.

Polyhydroxybutyrate, P(3HB), is the most well known and well characterized PHA.

However, industrial applications of P(3HB) have been hampered knowing to its low

thermal stability and excessive brittleness upon storage (Lee, 1996). The copolymer of

3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV), P(3HB-co-3HV), is more

flexible and tougher than the P(3HB). It can be used to make various products, including

films, coated paper, board, compost bags, disposable food service ware and moulded

products such as bottles and razors and also be used for biomedical applications (Lee,

1996).

Besides that, there have recently discovered 4-hydroxybutyrate, P(4HB). The

P(4HB), has been found to be useful in the biomedical applications (Martin and

Williams, 2003). It was used for tissue engineered heart valve scaffold and viable ovine

blood vessels (Chen and Wu, 2005). Also, a high molecular weight copolymer of 3HB

and 4HB [P(3HB-co-4HB)] containing 0–100 mol% of 4HB, can be produced by

Comamonas acidovorans with a controlled degradation rate (Saito and Doi, 1994),

making them ideal candidates for biomedical applications such as tissue engineering

(Martin and Williams, 2003). Other application of PHA are shown in Table 2.3.

Table 2.3: The possible application of PHA

• Packaging films, bags and containers

• Biodegradable carrier for long term dosage of drugs, medicines,

• insecticides, herbicides, or fertilizers

• Disposable items such as razors, utensils, diapers, or feminine

• hygiene products

• Surgical pins, sutures, staples, and swabs

• Wound dressing

13

2.10 Characterization and identification of bacteria

2.10.1 16S rRNA

Prokaryotic classification has historically relied on phenotypic attributes such as

size and shape, staining characteristics, and metabolic capabilities to group organisms.

Newer molecular techniques such as DNA sequencing give a greater insight into the

evolutionary relatedness of microorganisms. DNA sequences are viewed as evolutionary

chronometers, meaning that sequence differences appear to provide a relative measure of

the time elapsed since the organisms diverged from common ancestor (Willey, 2008).

DNA sequencing enables one to more accurately construct a phylogenetic tree.

These trees are somewhat like a family tree, tracing the evolutionary heritage of

organisms. Each line or branch of the tree represents the evolutionary distance between

two species. Individual species are represented as nodes.

2.10.2 Polymerase Chain Reaction

The first step is to synthesize DNA fragment with sequence identical to those

flanking the targeted sequence. This is accomplished with a DNA synthesizer. These

synthetic oligonucleotides are usually about 20 nucleotides long and serve as DNA

primer DNA synthesis. The primers are one component of the reaction mixture, which

also contains the target DNA, a thermostable DNA polymerase, and each of the four

deoxyribonucleoside triphosphates (dNTPs). PCR requires a series of repeated reactions,

called cycles. Each cycle has three step that are precisely executed in a machine called

thermocycler (Willey, 2008).

In the first step, the target DNA containing the sequence to be amplified is heat

denatured to make it single stranded. Next, the temperature is lowered so that the

14

primers can hydrogen bond or anneals to the DNA on the both sides of the target

sequence. Because the primers are very small and are present in excess, the targeted

DNA strands anneal to the primer rather than to each other. Finally, DNA polymerase

extends the primers and synthesizes copies of the target DNA sequence using dNTPs.

Only polymerase able to function at high temperatures employed in the PCR technique

can be used.

At the end of one cycle, the targeted sequences on both strands have been copied.

When the three step cycle is repeated, the two strands from the first cycle are copied to

produce four fragments. These are amplified in the third cycle to yield eight double-

stranded 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

19

3.2 Microorganisms

Eleven strains of bacteria coded strain 1, strain 2, strain 3, strain 5, strain 6, strain

8, strain 9, strain 11, strain 12, strain 13 and strain 14 were obtained from the culture

collection of Research Laboratory 2, Department of Biology, Faculty of Science,

Universiti Teknologi Malaysia, Skudai, Johor. The bacteria stored as glycerol stock

cultures at -20oC were revived by inoculating into universal bottles containing 5 mL of

sterile nutrient broth medium (Section 3.3.1) and incubated at 30oC for 24 h without

shaking. The purity of each bacterial culture was monitored by streaking onto separate

nutrient agar medium (Section 3.3.2) and incubated for 24 h at the same temperature.

The bacterial colonies grown on the solid medium were then observed under stereo

microscope (Leica model CME microscope). Culture observed with colonies of the same

morphology on nutrient agar is considered pure culture, whereas those observed with

two or more types of colonies are subjected to culture purification via single colony

isolation method. A single colony of different morphologies were aseptically selected,

streaked onto a separated Nutrient agar medium and allowed to grow at 30oC for 24 h.

Repeated single colony isolation was carried out until the culture purity was ensured.

3.2.1 Morphology characterization of bacteria

Pure cultures of bacteria were streaked onto Nutrient agar medium (Section

3.3.2) and incubated at 30oC for 24 h. The bacterial colonies formed on the surface of

the medium were then observed using a stereo microscope to record for their

morphology such as shape and pigmentation.

20

3.2.2 Preparation of inoculums

The pure bacterial strains were streaked onto the Nutrient agar medium (Section

3.3.2), incubated at 30oC for 24 h and pure colony was aseptically transferred into sterile

universal bottles containing 15 mL of the Nutrient broth medium (Section 3.3.1). The

bacteria were allowed to grow in a shaking incubator at 30oC, 200rpm for 24 h. The

culture’s turbidity was measured using a bench top spectrophotometer (Jenway 6300) at

the wavelength of 600nm. Cultures with the absorbance values ranging from 0.7-0.8

were used as inoculums.

3.2.3 Preparation of Stock Culture

Pure culture of bacteria were inoculated (10% v/v) into universal bottles

containing 10 mL of sterile Nutrient broth medium (Section 3.3.1) at 30oC for 24 h. A

0.8 mL of the fresh cultures were then transferred into a separate sterile Eppendorf tube

and added with 0.2 mL glycerol to a final volume of 15% v/v. The cultures were then

frozen in liquid nitrogen prior to place at -80oC freezer for long term storage.

3.3 Media preparation

Solid and liquid media used in this study are of enriched and defined types. All

media are prepared as described in section 3.3.1 to 3.3.3.

21

3.3.1 Nutrient Broth

This is an enrichment medium used to grow the bacteria when preparing the

inoculums. Nutrient broth has been a commonly used medium to grow heterotrophic

bacteria. In this study, the medium was used for the preparation of bacterial inoculums.

Nutrient broth consists of peptone, meat extract and distilled water. An 8 g/L of Nutrient

broth powder was added into a 1L Schott bottle containing 1L of distilled water. They

were mixed and sterilized via autoclaving at 121oC for 20 min. The broth was left to

cool to 50oC prior to pour into sterile universal bottle. The universal bottle can be

directly used or stored at 4oC for subsequent use.

3.3.2 Nutrient Agar

Nutrient agar is commercially obtained which consisted of peptone and beef

extract as the source of carbon, minerals and vitamins which are important to support

bacterial growth; sodium chloride as the carbon source of chloride ion and agar as

gelling agent. A 20.0 g/L of the nutrient powder was added into a 1L Schott bottle

containing 1L of distilled water. They were mixed and sterilized via autoclaving at

121oC for 20 min. The medium were left to cool at 50oC prior to pour into sterile Petri

plates. The agar was left to solidify at room temperature. The plates can be directly used

or stored at 4oC for later used. All plates were sealed with parafilm in order to avoid

contamination during storage.

3.3.3 Mineral Salt Medium

For PHA production from all the bacterial strain, the strain was grown in mineral

salt media containing 10 g/L glucose and microelement. The mineral salt media

consisted of (g/L) 0.5 (NH4)2SO4, 0.4 MgSO4.7H2O, 9.65 Na2HPO4.12H2O, 2.65

22

KH2PO4 in distilled water. All the content was autoclaved at 121oC for 20 min except

for MgSO4.7H2O. The MgSO4.7H2O was filter sterilized and added to the autoclave

mineral salt media. For the microelement, 1mL from the solution containing 20g

FeCl3.6H2O, 10g CaCl2.H2O, 0.03 CuSO4.5H2O, 0.05 MnCl2.4H2O and 0.1

ZnSO4.7H2O in 0.5M HCl was added to the mineral salt media.

3.4 Screening of PHA Producer

This screening method was used for detection of PHA production after

cultivation in mineral salt media. The purpose of this is to check or screen which

bacterial strain can produce high production of PHA.

Before continuing the screening, centrifuge tubes were first washed with ethanol

and hot chloroform to remove and substance or plasticizers. The cultures then were

centrifuged at 4000 x g for 30 minutes. The cell pellet or paste was suspended in a

volume of commercial sodium hypochlorite solution (Clorox) equal to original volume

of medium. After 1 h at 37oC, the lipid granule were centrifuged, wash with water and

then wash with alcohol and acetone. The polymer was dissolved with 5 mL of

chloroform and left overnight at 28oC on a shaker at 150rpm. Then the contents were

centrifuged at 4000 x g for 30 minutes. The supernatant was taken and transferred into

clean test tube. The supernatant containing chloroform is evaporated and 5 mL of

concentrated H2SO4 were added. The tube is capped and heated for 10 min at 100oC in

water bath. The solution is cooled to room temperature and the samples are transferred

to silica cuvette and the amount of PHA was determined at 235nm.

23

3.5 Characterization and identification of potential PHA producing bacteria

3.5.1 Biochemical Characterization

Enzymatic activities are widely used to differentiate bacteria. Even closely

related bacteria can usually be separated into distinct species by subjecting them to

biochemical tests, such as one to determine their ability to ferment an assortment of

selected carbohydrates. Moreover, biochemical tests can provide insight into a species’

niche in the ecosystem.

3.5.1.1 Catalase Test

This test is particularly useful in distinguishing staphylococci and micrococci,

which are catalase-positive, from streptococci and enterococci, which are catalase-

negative The test was done aseptically by picked up bacterial cell from colony of slant

growth with an inoculating loop. A drop of hydrogen peroxide was pipette onto the mass

of bacterial cells. The drops of hydrogen peroxide were observed to see if bubbles were

involved. The production of gaseous bubbles indicates the presence of catalase.

3.5.1.2 Cytochrome oxidase

The oxidase test is a test used in microbiology to determine if a bacterium

produces certain cytochrome c oxidases Strains may either be oxidase positive (OX+) or

negative (OX-). OX+ normally means that the bacterium contains cytochrome c oxidase

and can therefore utilize oxygen for energy production with an electron transfer chain.

A piece of filter paper was moistening in a Petri dish with a few drops of a

freshly prepared 1% (w/v) solution of tetramethy-p-phenylenediamine dihydrochloride.

A loopful of bacterial growth was aseptically transferred from agar medium and smear it

24

on the moisten paper. The development of violet or purple colour after observing the

filter paper within 10 seconds indicates a positive test.

3.5.1.3 Nitrate reduction

A nitrate test is a chemical test used to determine the presence of nitrate ion in

solution. The test was perform by inoculate bacterial cultures onto tubes containing beef

extract-peptone broth (Appendix D). The tubes were incubated at 37oC for 2-5 days. The

tubes were then observed and the displacement of the liquid is indicative of the

production of nitrogen. 2-3 drops of Reagent A (Appendix D) and 2-3 drops of Reagent

B (Appendix D) were added to each tube. Any immediate red colour demonstrates the

presence of nitrite and is indicative of reduction of nitrate and nitrite.

3.5.1.4 Citrate Test

Simmons citrate agar tests the ability of organisms to utilize citrate as a carbon

source. Simmons citrate agar contains sodium citrate as the sole source of carbon,

ammonium dihydrogen phosphate as the sole source of nitrogen and other nutrients.

Organisms which can utilize citrate as their sole carbon source use the enzyme citrase or

citrate-permease to transport the citrate into the cell. These organisms also convert the

ammonium dihydrogen phosphate to ammonia and ammonium hydroxide, which creates

an alkaline environment in the medium.

Using a sterile loop, bacterial culture was streak onto citrate slant agar (Appendix

G) and was labelled accordingly to each strain. All the tubes were then incubate at 37oC

for 4 days and observed any changes occurred.

25

3.5.1.5 Triple Sugar Iron Test

Triple sugar iron agar (TSI) is a differential medium that contains lactose,

sucrose, a small amount of glucose (dextrose), ferrous sulfate, and the pH indicator

phenol red. It is used to differentiate enteric based on the ability to reduce sulfur and

ferment carbohydrates.

A sterile inoculating loop with bacterial culture was streak onto TSI slant agar

(Appendix F). Using the same culture, another bacterial culture was stabbed onto

another TSI slant agar. The tubes were labelled carefully and incubate it at 37oC for 24

h.

3.5.1.6 Starch hydrolysis

The test involves the breakdown of starch into maltose. Firstly, all the bacterial

strain was inoculated by making a single streak down the middle of the plates. The

plates contained sterile nutrient agar supplemented with 0.2% (w/v) soluble starch. All

the plates were incubated at 37oC for 1 to 5 days. After incubation, the plates were

flooded with Lugol’s iodine (Appendix H). Any clear are indicates the hydrolysis of

starch and unchanged starch will stain dark blue.

3.5.1.7 OF-Glucose Test

OF medium is a nutrient semisolid agar containing high concentration of

carbohydrate and low concentration of peptone. The peptone will support growth of

bacteria that do not use carbohydrate. The test start with using a sterile needle and the

bacterial culture was inoculated each with two tubes of OF-glucose media (Appendix E).

2ml of sterile mineral oil was poured over one of the inoculating tube and replaced the

26

cap. The tube was labelled as anaerobic and the other (not added with mineral oil) as

aerobic tube. Both tubes were incubated at 37oC for 1 or 2 days.

3.5.1.8 Gelatin liquefaction Test

Many microorganisms produce gelatinase which catalyzes the hydrolysis of the

collagen. Bacterial culture was inoculated in the nutrient broth-gelatin tubes and all the

tubes was labelled and incubated at 37oC for 10-14 days. The tubes were placed in a

refrigerator for 30-60 minutes after incubation. Lack of gelatin hydrolysis will result in

liquid consistency of the medium.

3.5.1.9 Urease Test

Urease is an enzyme that catalyzes the hydrolysis of urea, forming ammonia and

carbon dioxide. It is found in large quantities in jack beans, soybeans, and other plant

seeds, it also occurs in some animal tissues and intestinal microorganisms.

The surface of the Christensen urea agar slant (Appendix I) was inoculated with

bacterial strain and was labelled accordingly. All the tubes were incubated at 37oC for 1-

5 days. After the incubation, the appearance of a red violet colour indicates a positive

test while a yellow orange colour indicates negative result.

3.5.1.10 Indole Test

Tryptone broth (Appendix 10) tubes were inoculated with bacterial strain. All the

tube then incubated at 37oC for 24 to 48 hours. 10 drops of Kovac’s reagent (Appendix

27

H) were carefully layered directly onto the top of the broth culture tube. An immediate

formation of a red layer at the top of the broth indicates the presence of indole and a

yellow or brown colour is a negative test.

3.5.1.11 Motality

Motality test are used to identify the organism or bacteria that are able to move.

In this test, positive test indicates the organism is motile which cause turbidity in the

medium.

The media contain beef extract (3.0g), peptone (10.0g), NaCl (5.0g) and Agar

(4.0g) in 1L of distilled water. The media was autoclaving at 121oC for 15 minutes.

Pure culture was stabbed into the medium with a sterile needle to a depth of 1 inch and

incubated at 37oC for 1-2 days

3.5.1.13 Lipase Test

The purpose for Lipase test is to determine whether a bacterium produces a

lipase that will hydrolyze a neutral fat to fatty acid and glycerol. The medium used for

the test is Spirit Blue agar (Appendix K).

3.5.1.14 Voges Proskauer Test

The principle for this test is to detect the production of acetylmethylcarbinol, a

natural product formed from pyruvic acid in the course of glucose fermentation. Positive

result show pink colour after added α-napthol, and 1 mL of 40% KOH.

28

3.5.2 Molecular Characterization

3.5.2.1 DNA Extraction

A 5 ml of overnight culture was added into a centrifuged tube. The tube was

centrifuged at 13,000 rpm for 3 minutes. The supernatant were removed and discarded.

Then, the pellet was resuspended in 480µl of 0.5M EDTA. 120µl of lysozyme was

added gently mixing by inverting the centrifuge tube and later incubated it at 37oC for 1

hour. After 1 hour, the culture was centrifudge at 13,000 rpm for 3 minutes. The

supernatant was removed and 600µl of nucleic lysis solution was added and mix gently

until the cell resuspended. The suspension then incubated at 80oC for 5 minutes to lyse

the cell and let it cool to room temperature. 3µl of RNase solution was added to the cell

lysate and let it mix by inverting the tube for several time. The mixture was then

incubated at 37oC for 30 minutes and let it cool to room temperature. A 200µl of protein

precipitation solution was added to the RNase-treated cell lysate and vortexing it at high

speed. Further incubation was perform for the culture on ice for 5 minutes and

centrifuged at 13,000 rpm for 3 minutes. The supernatant was transferred into a fresh

centrifuged tube which contains 600µl isopropanol (at room temperature) and gently

mix it by inverting the tube. The tube was centrifuged at 13,000 rpm for 5 minutes. The

supernatant was carefully poured and drain the tube on clean absorbent paper. 600µl of

70% (v/v) ethanol was added and gently mix it to wash the DNA pellet and followed by

centrifuged the tube at 13,000 for 3 minutes. The supernatant was carefully drain and air

dried for 10-15 minutes. 100µl of DNA rehydration solution was added to the tube and

incubated overnight at 4oC.

The success of genomic DNA isolation was determined by agarose gel

electrophoresis analysis.

29

3.5.2.2 PCR Amplification of 16S rDNA Fragment

PCR Amplification of 16S rDNA was carried out using universal primers pA or

fd1-07 (forward) and pH or rd1-07 (reverse). The sequences of respective primer are

shown in table 3.1. To start the PCR, annealing temperature (Ta) of the primer must be

known. Usually, the annealing temperature starts at 5oC below the calculated

temperature of primer melting point (Tm).

Ta = Tm - 5oC

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

Table 3.1 : Sequences of eubacterial 16S rDNA universal primers

Primers Sequence

pA 5’- AGA GTT TGA TCC TGG CTC AG -3’

pH 5’- AAG GAG GTG ATC CAG CCG CA -3’

Mixture to run the PCR contains PCR Master Mix (Promega), forward and

reverse primer, genomic DNA template and nuclease free water. To prepare, genomic

DNA template was added accordingly to the concentration of the genomic DNA, primer,

PCR Master Mix and nuclease free water was added to the total of 50 µL of all the

mixture.

30

Table 3.2 : PCR reaction mixtures

Volumes (µL) Reagents

Strain 2 Strain 5 Final Concentration

DNA Template 1 3 < 250 ng

Forward Primer 1 1 0.1 – 1.0 µM

Reverse Primer 1 1 0.1 – 1.0 µM

2X PCR Master Mix 25 25 1X

Nuclease Free Water 22 20 -

Total 50 50 -

Table 3.3 : Gradient PCR cycle profile

Steps Temperature Duration

Initial Denaturation 94oC 4 min

Denaturation 94oC 1 min

Annealing 50 – 55oC 45 seconds

Extension 72oC 1 min

Final Extension 72oC 7 min

3.5.2.3 Purification of the Amplified 16S rDNA Fragment

Purification of the amplified 16S rDNA was done by using Wizard® SV Gel &

PCR Clean-up System (Promega) according to the manufacturer’s instructions. The

purpose for doing the purification is to remove enzymes, ethedium bromide, mineral oil,

agarose, nucleotides, primers, salts, and other impurities from the DNA samples.

Purication method can be done by using two methods; gel dissolving and direct

PCR product solution.

31

3.5.2.4 Agarose Gel Electrophoresis

First step in doing agarose gel electrophoresis is to prepare the agarose gel. 1g of

agarose was weight out and added into 50ml 1XTAE buffer. The suspension was heated

in a microwave until dissolved. The solution was left to cool at about 40oC and ethidium

bromide was added into the molten agarose solution. Then, the molten agarose was

added into gel molding tray and carefully inserted a comb into the molten gel to allow

the formation of well for DNA loading. Carefully, the combs were removed by pulling

them upwards firmly and smoothly in a continuous motion.

After finished preparing the gel, the next step is to prepare the DNA sample for

electrophoresis. Firstly, a 5µl of the DNA sample was aliquot into a microcentrifuged

tube. 1µl of loading dye was added and mix it by flicking the tube several times. The

mixture was loaded into the well. A 10kb mass ruler DNA marker was used as standard

for determining the size of DNA fragment. The electrophoresis chamber was closed with

lid and connects all power cable according to the colour code. The gel was run at 80V

for approximately 1 hour and observed.

3.5.2.5 Sequencing of the Amplified 16S rDNA Fragment

The PCR product that have been purified, were sent to 1st BASE Laboratory Sdn.

Bhd., Selangor. Both forward and reverse primer was also included as sequencing probe.

3.5.2.6 Phylogenetic Tree Construction

Sequence result obtained from 1st BASE Laboratory Sdn. Bhd., Selangor, were

analyzed using online software BLASTn (Basic Local Alignment Search Tool of

nucleotide). The sequence match obtain from the BLASTn are subjected to ClustalX

32

program to align the multiple sequence. It calculates the best match for selected

sequence and lines them up so that the identities, similarities and differences can be

seen. Evolutionary relationships can be seen via tree view application.

3.6 Analytical Methods

3.6.1 Determination of PHA

PHA that was extracted from cell was determined by using UV-

Spectrophotometer. Before the determination, the solution (Section 3.4) was diluted with

sterile distiller water. The solution was diluted to the dilution factor of 10. This is

because, the initial solution contain concentrated sulphuric acid and cannot be read by

the UV-Spectrophotometer.

The absorbance of the UV-Spectrophotometer was set to 235nm and all the

absorbance reading of each samples was recorded and check.

3.6.2 Determination of Bacterial Growth

The normal bacterial growth curve has four phases; the lag phase, the log phase,

the stationary phase and death phase. This curve is affected by environmental and

nutritional factors.

45 mL of nutrient broth with 5 mL of bacterial inoculums was inoculated in

conical flask at put in shaker incubator at 200rpm and the temperature was set at 30oC.

The time interval to measure the optical densities is every 30 minutes. 1 mL from each

flask was transferred to cleaned cuvettes to check the optical densities by using

spectrophotometer at 600nm.

CHAPTER 4

RESULTS AND DISCUSSIONS

4.1 Screening of potential PHA producer

Some bacteria have been identified that have the ability to produce PHA. The

productions of PHA are depended on the environmental factor, growth condition and the

nutrient available. Referring to Wennan He, (1998), PHA production can be enhanced

by using mineral salt media. The medium used to grow the bacterial strains can affect

the production of PHA from bacteria. By using mineral salt media, the PHA production

was successfully produced from the bacteria. The mineral salt media are supplemented

with glucose as the carbon source. PHA was produced by the bacteria intracellulaly.

During the cultivation of the bacteria, some of the bacterial strain produced slimy and

foam. Different kinds of smell were also produced.

The PHA produced in the cell were harvested by using dispersion of chloroform

and aqueous sodium hypochloride. Before the harvesting of PHA, all the centrifuged

tubes were washed with ethanol and hot chloroform. This is to ensure the removal of any

plastic material that maybe presence in the centrifuged tubes than can be interfering with

the results of screening for PHA production from the cell.

During the extraction of the PHA from the cultivated bacterial strains, there are

some precaution procedures that must be taken seriously. Because the extraction used

solution that are dangerous which are some volatile and corrosive, the use of lab coat,

latex glove and mouth mask must be used. Some solution such as hot chloroform,

ethanol, and concentrated sulfuric acid must be handling with care.

34

The addition of sulphuric acid in the last step of extraction of the PHA is very

concentrated and it cannot be read by UV-Spectrophotometer at 235nm. So, the

concentrated sulfuric acid that contains PHA must be diluted first. Silica cuvette was

used because the normal cuvette can react with the sulfuric acid and can interfere with

the reading of UV-Spectrophotometer.

Figure 4.1 Amount of PHA produce by bacterial strains

35

The amount of PHA extracted was determined as the following (Slepecky and Law, 1960).

A = kbc

OD = 1.55 x 10-4 x 1 x c x DF

c = OD x DF 1.55 x 104 x 1 A = optical density (OD)

k = molar extinction coefficient of crotonic acid

b = diameter of cuvette

c = concentration of PHA

DF = dilution factor

By using the formula, results showed in Figure 4.1 indicated that strains 1, 2, 5

and 6 were among the potential PHA producers. However, two strains which are strain 2

and strain 5 showed highest production of PHA compared to other strains. This could

possibly due to the ability of these strain to metabolized glucose more efficiently and

resulted to the production of high biomass for the accumulation of PHA. Strain 8, strain

12 and strain 13 showed low production of PHA. This maybe due to the limited amount

of cell or they may favor other environmental factor and other carbon source besides

glucose. Based on the report by Wennan He, (1998), glucose was also found to be the

favorable substrate (carbon source) for PHA production.

4.2 Colony and Cellular Morphologies Characterization

A total of 11 strain of pure colony of bacteria was successfully grow from the

culture collection of Research Laboratory 2, Department of Biology, Faculty of Science,

Universiti Teknologi Malaysia, Skudai, Johor. All the strains of pure colony show

different characteristic in morphologies and sizes.

36

Some of the strains grow very turbid and produce slime which is called EPS.

EPS is an extracellular polysaccharide which enables a bacterium to survive by attaching

to various surfaces in its natural environment in order to survive. Through attachment,

bacteria can grow on diverse surfaces such as rocks, plant roots and even on bacteria.

These attachments are due to the present of glycocalyx. Besides giving attachment, the

glycocalyx can also protect a cell against dehydration. For example, strain 8 (Figure 4.7)

and strain 13 (Figure 4.11).

Strain 2 and strain 3 show similarity in the shape of colony. However, strain 2

grow faster and have a clear zone at the end of the colony and differ to strain 3. Many of

the strains show same pigmentation colour which is white (strain 1, strain 2, strain 3,

strain 5, strain 6, strain 8 and strain 13), yellow (strain 9, strain 11 and strain 12) and

pink (strain 14).

Most of the strains appeared to have rough surface due to the characteristic of the

strains and colony growth. However, for strain 8 and strain 13, the surface of the colony

appeared to be smooth due its slimy growth. This could lead to inability for the strains to

produce single colony.

37

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

Strain Forms Elevation Margins Colony

surface Pigmentation

Optical

Characterization

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

38

Figure 4.2 Colony morphology Strain 1 Figure 4.3 Colony morphology Strain 2



39



Figure 4.12 Colony morphology Strain 14

40

4.2.1 Gram Staining

Gram staining was done to further characterize the pure colony of the bacterial

strains to observe their cellular morphology and arrangement. Strain 2 and strain 5 was

selected to be characterize because its ability to produce high amount of PHA. The

cellular characteristics of the strains are shown in Table 4.2.

Table 4.2: Cellular characterization of strain 2 and strain 5

Strain Gram Reaction Cellular Arrangement Possible related Microbes

2 Figure 4.27

Positive Rod shape Bacillus species

5 Figure 4.28

Negative Branching coccobacilli (oval and cocci)

Under phylum Acinetobacter

Under the microscope, the gram staining results revealed that strain 2 appeared to

be gram positive. Its cellular arrangement is rod shape. It can be possibly related to

Bacillus species. Meanwhile, strain 5 appeared to be gram negative. Its cellular

arrangement is similar to branching coccobacilli which is oval and cocci in shape. It can

be possibly related under phylum Acinetobacter.

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

41

4.3 Biochemical Characterization

Biochemical test are used to determine and to differentiated the bacteria based on

their properties. The biochemical tests were catalase, cytochrome oxidase, nitrate

reduction, citrate, TSI, starch hydrolysis, OF-Glucose, gelatin liquefaction, urease,

indole, lipase, gram staining, MacConkey agar, Voges-proskauer and motility test. The

results are summarized in the Table 4.3.

Table 4.3 : Summary of Biochemical test result

Biochemical Test Strain 2 Strain 5

Catalase - +

Urease - +

Citrate - -

Nitrate + +

Cytochrome Oxidase - -

Lipase + +

Gel Liquefaction - -

Indole - -

Starch - +

Voges - -

Motality - -

Mcconkey - +

Gram Staining + -

TSI Fermentation of glucose and

lactose Fermentation of glucose

and lactose

OF-Glucose Fermentative organism Fermentative organism

Table 4.3 summaries the biochemical test that have been done on strain 2 and

strain 5. From the catalase test, strain 2 show no reaction which give negative result and

42

strain 5 show reaction with production of bubbles which give positive result. Catalase is

an enzyme that catalyzes the decomposition of hydrogen peroxide (H2O2) to water and

gaseous oxygen. Most aerobic microorganisms possess catalase. The function of catalase

is to remove toxic hydrogen peroxide that forms during the oxidation-reductions

reactions that are coupled with oxygen in respiratory metabolism. The presence of

catalase is shown when hydrogen peroxide is added to a colony or loopful of bacteria

and bubbles of oxygen are released from the surface (Jean F, 1980).

For the nitrate reduction test, both of strain 2 and strain 5 showed positive result

(Figure 4.15, Figure 4.16). The strain can use nitrate as the terminal electron acceptor in

place of oxygen during respiratory metabolism. When this occurs, the pathway is called

anaerobic respiration. In anaerobic respiration using nitrate as the terminal electron

acceptor, nitratase catalyzes the reduction of nitrate to nitrite. The nitrate may be further

reduced to nitrogen gas (Jean F, 1980). The presence of nitrogen gas release from the

strains is showed by red color formation.

Figure 4.15 Nitrate reduction test of Figure 4.16 Nitrate reduction test of

Strain 2 Strain 5

Strain 2 and strain 5 did not show any positive result to the citrate test. This is

because the strains do not utilize citrate as the sole carbon source of carbon. The

organisms that utilize citrate and produce an alkaline reaction as indicated by the

bromothymol blue, which changes the color from green to blue (Jean F, 1980). Positive

result for citrate test is shown by turbidity and blue color changes from green color

medium.

43

Triple sugar ion (TSI) is carried out to determine the ability of an organism to

attack a specific carbohydrate incorporated in a basal growth medium, with or without

the production of gas, along with the possible hydrogen sulfide (H2S) production. Strain

2 and strain 5 show positive results for the fermentation of glucose and lactose (Jean F,

1980).

Starch is a homopolysaccharide, a condensation product of many monomers of a

single type of monosaccharide, α-D-glucose, forming a polymer of many units united by

α-glucosidic linkages. Starch hydrolysis test is to check the presence of extracellular

amylolitic enzymes that breakdown starch. Strain 2 (Figure 4.17), show negative results

for starch hydrolysis test and this showed that it cannot breakdown the starch. Strain 5

(Figure 4.18) showed positive result and was able to breakdown the starch. Positive

results on the medium are shown by purple-blue with colorless area around growth of

bacteria (Jean F, 1980). Advantage of strain 5 being able to hydrolyze starch are

important to producing PHA as it can act to provide the need of other strain that lack this

ability.

Figure 4.17 Starch hydrolysis test Figure 4.18 Starch hydrolysis test on strain 2 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. Fermentation is an aerobic process and bacterial fermenters of

a carbohydrate are usually facultative anaerobes. By the fermentation process, a

44

carbohydrate is metabolized and split into two triose carbon molecules (Jean F, 1980).

Strain 2 and strain 5 are both fermentative bacteria by the result of OF-Glucose test.

Yellow color formation on both medium confirmed the results.

Urease test on strain 2 (Figure 4.19), gave negative results howeverpositive for

strain 5 (Figure 4.20), wioth red color changes of the medium. The enzyme urease,

catalyze the breakdown of urea into ammonia and carbon dioxide. Microorganisms that

produce this enzyme are able to detoxify a waste product and derive metabolic energy

from its utilization

Figure 4.19 Negative result of Figure 4.20 Positive result of urease

urease test of strain 2 test of strain 5

Strain 2 and strain 5 showed positive result of lipase activity. Lipase test are used

to determine whether a bacterium produces a lipase that will hydrolyze a neutral fat to

fatty acid and glycerol.

Gelatin liquefaction test showed positive result for both strain 2 and strain 5. The

strains possess gelatinase which is produced to catalyze the hydrolysis of the protein

gelatin (collagen). The hydrolysis of gelatin produces soluble carbohydrates that are

readily metabolized as a source of carbon source.

.

45

Figure 4.21 Gel liquefaction test Figure 4.22 Gel liquefaction test of strain 2 of strain 5

Voges Proskauer test is used to determine the ability of organisms to produce a

neutral end product, acetylmethylcarbinol (acetoin), from glucose fermentation. Both

strain 2 and strain 5 lacks this ability. Glucose is metabolized to pyruvic acid which is

the key intermediate in glycolysis. From pyrivic acid there are many pathways a

bacterium may follow. The production of acetoin is one pathway for glucose degradation

occurring in bacteria.

The ability to hydrolyze tryptophan to indole is a characteristic of strain enteric

bacteria that possess the enzyme trytophanase. Trytophanase catalyze the hydrolysis of

tryptophan with the production of indole, pyruvic acid and water. However, strain 2 and

strain 5 did not have this ability and thus give negative result in the indole test.

Strain 2 and strain 5 are nonmotile bacteria from the motality test. The purpose

for motality test is to determine if an organism is motile or nonmotile. Bacteria are

motile by means of flagella. Flagella occur primarily among the bacilli. However, a few

coccal forms are motile. Motile bacteria may contain a single flagellum or many flagella.

Nonmotile organisms lack flagella.

MacConkey Agar and gram staining is a test that determined and differentiated

between gram positive and gram negative bacteria. Growth on MacConkey agar

indicates that the bacteria are gram negative bacteria. Strain 5 (Figure 4.24), is gram

negative bacteria by means of growth on MacConkey agar and gram staining.

46

Figure 4.23 No growth on MacConkey Figure 4.24 Growth on MacConkey

agar of strain 2 agar of strain 5

From the overall biochemical test result, Its showed that strain 2 are related to

genus Bacillus species and strain 5 are related under phylum Acinetobacter.

4.4 Bacterial growth analysis

Bacterial growth refers to an increase in bacterial numbers, not an increase in the

size of the individual cells. Bacteria normally reproduce by binary fission. Binary fission

is a prokaryotic cell reproduction by division into two daughter cells. Once cell division

begins, it proceeds exponentially with one cell dividing to form two, each of these cells

dividing so that four cell form, and so forth in geometric progression.

From Figure 4.25, Strain 2 and strain 5 shows normal growth curve of bacteria.

The curves consist of 4 phases which is the lag phase, the log phase, the stationary phase

and the death phase. Stationary phase for strain 2 are shorter in time compare to strain 5

but the time for lag and log phase for strain 5 are faster than strain 2.

Referring to the cell dry weight analysis, PHA probably being produce at the log

phase and as time passed, the availabilty of nutient and biomass accumulated end or less

when the phase started to enter stationary phase. The strains started to enter the death

phase because nutrient that are needed for the production of PHA by the strains are

depleted and the number of deaths exceeds the number of new cells formed.

47

From the ln x analysis against time for strain 2 and strain 5, specific growth rate

(µ) for strain 2 is 0.520 (Appendix 2) and specific growth rate for strain 5 is 0.576

(Appendix 2). From the value, its shows that strain 5 can grow faster than strain 2.

Figure 4.25 Cell dry weight analysis plot of strain 2 and strain 5

4.5 16S rRNA Sequencing

4.5.1 Genomic DNA extraction

Chromosomal DNA of strain 2 and strain 3 were extracted using Promega

WizardTM Genomic DNA Purification Kit. The Kit is an easy and reliable method to

purify gram positive bacteria genomic DNA. It is an advanced method compare to the

conventional method that use many steps that involve the use of organic solvent.

The kit consist of EDTA, lysozyme, Nucleic Lysis Solution, RNase solution,

isopropanol, ethanol, DNA Rehydration solution and protein precipitation solution.

48

Lysozyme was used to weaken the cell wall by breaking the β-1.4 bonds of

peptidoglycan bond. The addition of RNAse was to remove RNA that contaminating the

genomic DNA extract. The use of Nucleic Lysis solution is to weaken and lyse the

nucleic acid. Protein Precipitation solution was added to remove protein but leaves the

high molecular weight genomic DNA in solution. Isopropanol and ethanol would

precipitate and concentrate the DNA. DNA Rehydration Solution was added to

rehydrate the DNA and was stored at -20oC to prevent from contamination and

degradation of the DNA.

Agarose gel electrophoresis was used to analyze the isolated genomic DNA to

determine the success of the isolation (Figure 4.26). A clear and visible DNA band that

showed in lane 1 and 3 indicated that the isolation of genomic DNA of strain 2 and

strain 5 were done successfully. The bands that present indicate that the DNA isolated

were more than 10,000 bp in size. The visibility of the band obtained shown that the

genomic DNA was pure enough for sebsequent PCR amplification purpose. First

replicate of strain 2 and first replicate of strain 5 were selected for PCR amplification.

Figure 4.26 Agarose gel analysis of genomic DNA (1% w/v agarose, 80 volts, 45

watts, 60 minutes)

DNA Marker: MassRulerTM DNA Ladder

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

10000bp

4 3 1 2

49

Purity and concentration of genomic DNA was determined

spectrophotometrically by using a Varian Cary 100 model UV-Vis Spectrophotometer.

The concentration of the DNA was determined at the absorbance of 260nm (A260)

whereby an optical density (OD) of 1 corresponding to approximately 50 ng/mL of the

double stranded DNA. To calculate the purity of genomic DNA, ratio between A260 and

A280 (A260/A280) was calculated. The readings of 1.7-2.0 indicate that the genomic DNA is

highly purify (Adams, 2003). The ratio (A260/A280) less than 1.7 is considered not pure

enough and indicated to the presence of contamination such as protein and phenol. Table

4.4 summarizes the quantitative analysis of genomic DNA.

Table 4.4 : Quantitative analysis of mixed culture genomic DNA

Replicate A260 A280 A260/280 Dilution factor (DF)

A260 x DF

Concentration (µg/µl)

Strain 2 (1)

0.02686 0.01640 1.64 103 26.86 1.343

Strain 5 (1)

0.01167 0.00966 1.20 103 11.67 0.584

From Table 4.4, result show that the genomic DNA was not pure enough for

strain 2 and strain 5 as the ration of A260/A280 was not within 1.7 – 2.0. This could be

cause by contamination that presence in the genomic DNA. The concentration of strain 2

and strain 5 are also low but the PCR amplication were carried out and successfully

manage to get the PCR product.

4.5.2 PCR amplication of 16s rRNA gene fragment

Amplifications of the DNA samples were carried out using pA and pH primer.

The annealing temperature (Ta) for the primer is 55oC. Based on the genomic isolation, 1

µg/µL of template DNA from strain 2 and 3 µg/µL of template DNA was used to start of

the PCR reaction. Band on the agarose gel was shown in Figure 4.13 indicated with four

50

clear and strong bands with approximately 1500 bp in length were observed. This

indicated that the region of 16S rRNA was successfully amplified using pA and pH

primers.

Figure 4.27 Agarose gel analysis of PCR products (1% w/v agarose, 80 volts, 45

watts, 60 minutes)

4.5.3 Purification and qualitative PCR product analysis

Purification of the PCR reaction was done using the Wizard® SV Gel & PCR

Clean-Up System (Promega). Figure 4.14 show that the success of PCR products from

as visualized under UV lamp. The band showed on the agarose gel appeared to be

concentrated however replicate one of strain 2 did not show any visible band. This

maybe becaused by the lost during the purification step.

DNA Marker: MassRulerTM DNA

Ladder Mix, 10kbp

(Promega)

Lane 1: 1st replicate of strain 2

Lane 2: 2nd replicate of strain 2



1500 bp

1 2 3 4

51

Figure 4.28 Agarose gel electrophoresis of the purified PCR products (1% w/v

agarose, 80 volts, 45 watts, 60 minutes)

4.5.4 DNA sequence analysis The result of purified PCR product for strain 2 and strain 5 after sequencing

shows the appearences of multi N-terminal within the sequences. This result occurs

because the PCR product that was sent is low in purity and also the concentration.

4.5.4.1 BLASTn analysis BLASTn performed pairwise comparison of DNA sequences and align to

determine the homology of the query sequence. The blasting result for strain 2 showed

100 bases quried. The best scores gives similarity of 82% and this suggested that the

bacteria might be closely related to genus Bacillus species. However, strain 5 did not

produce any result might due to the sequence result of the purified product.

DNA Marker: MassRulerTM DNA

Ladder Mix, 10kbp

(Promega)





1500 bp

1 2 4 3

52

4.5.4.2 ClustalX After the BLASTn, the sequences were subjected to ClustalX program. ClustalX

program combines a good hierarchical method for multiple sequence alignment with an

easy to use interface and for preparing phylogenetic tree. Phylogenetic tree is a graphical

representation of the evolutionary relationship among a group of organisms or genes. To

view the phylogenetic tree produce after running ClustalX analysis of the sequence, Tree

View was used. From Figure 4.29, tree showed that strain 2 might be related to Bacillus

species that are known can be producing PHA.

Clostridium perfringens ABOO01

Strain 2

Bacillus thuringiensis EF63321

Pseudomonas sp EU557337

Bacillus sp EF633269

Bacillus cereus EF633204

Figure 4.29 Phylogenetic tree processed and illustrated by Tree View

CHAPTER 5

CONCLUSION AND FUTURE WORK

5.1 Conclusion

The efficiency of PHA produce by bacteria depends on the species and how the

nutrient is given to the bacteria. Different species of bacteria can be identified from the

identification of the shape, color and sizes of the colony produce. Plastics produced from

bacteria have become a possible solution to dumping waste because the plastics can

breakdown easily as compared to the conventional plastics that were produced from

petroleum based compounds.

Using minimal salt media with the supplementation of glucose as carbon source,

bacterial strains 2 and 5 produce similar amount of PHA of 1.49 mg/L respectively.

Although other strains screened were able to produce PHA, the amount were very low

compared to strain 2 and strain 5. The bacterial growth of strain 2 and strain 5 were

almost the same but the growth rate of strain 5 appeared to be faster than strain 2.

From the biochemical testing and gram staining results, strain 2 and strain 5 are

from Bacillus species and from phylum acinetobacter respectively. Molecular

identification supports the result of strain 2. The biochemical tests are able to

differentiate the two strains by several tests that have been done.

54

5.2 Future Work

There are several improvements and further study can be suggested:

1. Using several other carbon source and environmental factor such as pH,

temperature and availability of oxygen. These factors can be manipulated to

check whether the production can be enhanced and high productivity can be

accumulated.

2. Genetic engineering method can be introduced in order to higher the limits of

production of PHA. The gene responsible for the production can be identified

and further studied.

3. Besides researching for production of PHA by bacteria, study can also conducted

on the effectiveness of the degradability function of the PHA. PHA can be

degraded by nature but time for the degradation can also be account as it can be

positive or negative effect on the environment.

4. In addition, the use of DGGE or denaturing gradient gel electrophoresis can be

introduced as it can be used as a tool of genetic fingerprinting for a purpose to

investigate the diversity of microbial community in specific niches. Using

DGGE, a mixture of 16S rRNA fragments with different sequences will resolve

into a distinct pattern of bands. PCR and DGGE studies have confirmed that

standard culture techniques can be poor indicators of the composition of natural

microbial populations.

55

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60

APPENDICES

APPENDIX A Table : OD at 600nm analysis, cell dry weight and ln X value of strain 2 and strain 5

OD (600nm) X (mg/mL) ln X Time Interval (h)

Strain 2 Strain 5 Strain 2 Strain 5 Strain 2 Strain 5

0.0 0.028 0.209 0.28 2.09 -1.27 0.74

0.5 0.018 0.178 0.18 1.78 -1.71 0.58

1.0 0.031 0.224 0.31 2.24 -1.17 0.81

1.5 0.027 0.250 0.27 2.50 -1.31 0.92

2.0 0.044 0.243 0.44 2.43 -0.82 0.89

2.5 0.036 0.378 0.36 3.78 -1.02 1.33

3.0 0.048 0.521 0.48 5.21 -0.73 1.65

3.5 0.093 0.730 0.93 7.30 -0.07 1.99

4.0 0.122 0.816 1.22 8.16 0.20 2.10

4.5 0.230 1.165 2.30 11.65 0.83 2.46

5.0 0.365 1.343 3.65 13.43 1.29 2.60

5.5 0.761 1.511 7.61 15.11 2.03 2.72

6.0 1.305 1.787 13.05 17.87 2.57 2.88

6.5 1.949 7.310 19.49 73.10 2.97 4.29

7.0 5.620 6.980 56.20 69.80 4.03 4.24

7.5 5.960 7.050 59.60 70.50 4.09 4.26

8.0 8.130 8.770 81.30 87.70 4.39 4.47

8.5 8.150 8.400 81.50 84.00 4.40 4.43

9.0 8.100 8.460 81.00 84.60 4.39 4.44

61

9.5 8.150 8.600 81.50 86.00 4.40 4.45

10.0 8.110 8.610 81.10 86.10 4.40 4.46

10.5 7.690 8.450 76.90 84.50 4.34 4.44

11.0 7.590 8.440 75.90 84.40 4.33 4.44

11.5 6.970 8.430 69.70 84.30 4.24 4.43

12.0 8.160 81.60 4.40

12.5 8.100 81.00 4.39

13 7.780 77.80 4.35

13.5 7.340 74.30 4.31

62

APPENDIX B

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

Figure ln X analysis plot of strain 2

Figure ln X analysis plot of strain 5

63

APPENDIX C

Table : Value of OD and amount of PHA produce by bacterial strains

Bacteria OD Value Amount of PHA (mg/L)

Strain 1 10.7455 0.6934

Strain 2 23.1087 1.4909

Strain 3 6.01330 0.3879

Strain 5 23.1496 1.4935

Strain 6 10.3235 0.6603

Strain 8 4.36460 0.2816

Strain 9 6.97910 0.4503

Strain 11 4.90250 0.3163

Strain 12 4.41500 0.2848

Strain 13 3.18360 0.2054

Strain 14 9.42380 0.6079

64

APPENDIX D

1. Beef extract peptone broth:

Composition Amount (g/L)

Beef extract 3.0

Peptone 5.0

Potassium nitrate 1.0

2. Reagent A: 0.8% solution of sulfanic acid in 5N acetic acid.

3. Reagent B: 0.5% solution of dimethyl-α-naphthylamine in 5N acetic acid

*Possible and can be potentially carcinogen.

65

APPENDIX E

Preparation of OF-Glucose Medium


Peptone (pancreatic digest of casein) 2.0

Sodium chloride 5.0

di-Potassium hydrogen phosphate anhydrous 0.3

Agar 2.5

Bromothymol blue (1.5% w/v stock)

Glucose (10% w/v stock)

Method:

1. All the chemical compositions were mixed in distilled water and stirred it to

dissolve it. pH was adjusted to 7.0.

2. 3 mL of bromothymol blue was added to the medium.

3. 50 mL of the medium was autoclaved at 121oC for 15 minutes.

4. 5 mL of filter sterilize glucose was added aseptically from the stock solution

of glucose.

5. Later, 5 mL of the medium was dispensed in sterile test tubes.

66

APPENDIX F

Preparation of Triple Sugar Iron (TSI) Agar


Beef extract 3.0

Yeast extract 3.0

Peptone 20.0

Lactose 10.0

Sucrose 10.0

Glucose 1.0

Ferrous sulphate, FeSO4 0.2

Sodium thiosulphate, Na2S2O3 0.2

Sodium chloride, NaCl 5.0

Agar 12.0

Phenol red 0.024

Method:

1. All the chemical compositions were dissolved into 100 mL of distilled water

and pH was adjusted to 7.4.

2. The medium was then poured into test tubes each containing approximately 5

mL to make long slant agar.

3. All the tubes were then autoclaved at 121oC for 15 minutes and cooled to

room temperature in slanted position.

67

APPENDIX G

Preparation of Simmons Citrate Agar


Ammonium dehydrogen phosphate 1.0

Sodium ammonium phosphate 1.0

Sodium citrate 2.0

Magnesium sulphate 0.2

Sodium chloride 5.0

Alcoholic solution bromothymol blue (1.5% w/v) 10.0

Agar 20.0

Method:

1. All the chemical composition was dissolved into 1000 mL of distilled water

and the pH was adjusted to 7.4.

2. The medium was then poured into test tubes each containing approximately 5

mL to make long slant agar.

3. All the tubes were then autoclaved at 121oC for 15 minutes and cooled to

room temperature in slanted position.

68

APPENDIX H

Preparation of Lugol’s Iodine

Method:

1. 1.0 g of potassium iodide (KI) was dissolved in 100 mL distilled water.

2. Then, 5.0 g of crystal iodine (I2) was added slowly and shake it until

dissolved in the mixture.

3. Finally, the solution was filtered and stored in brown bottle

Preparation of Kovac’s Reagent

Method:

1. A 10 g of p-dimethylaminoensaldehyde dissolved in 150 mL of pure isoamyl

alcohol.

2. A concentrated HCl is then slowly added into the aldehyde-alcohol mixture.

69

APPENDIX I

Preparation of Christensen urea agar slant


Monopotassium 2.0

Sodium chloride 5.0

Peptone 1.0

Glucose 1.0

Urea 20.0

Phenol red 0.01

Agar 15

Method:

1. All the compositions were dissolved in 900 mL of distilled water except

urea and autoclaved it at 121oC for 15 minutes.

2. 20 g urea and 1 g glucose were rehydrated in 100 mL distilled water and

filter sterilized it using syringe.

3. The urea and glucose solution that have been filtered was added aseptically

into the sterile medium.

4. The medium were then poured into test tubes each containing

approximately 5 mL and solidify it in slanted position.

70

APPENDIX J

Preparation of Tryptone Broth medium


Tryptophan 2.0

Yeast extract 3.0

Sodium chloride 5.0

di-Sodium hydrogen phosphate 1.0

Method:

1. All the chemical composition above was dissolved in 1000 mL of distilled

water and the pH was adjusted to 6.8.

2. The medium were then poured into test tubes each containing approximately

4 mL.

3. The medium was then autoclaved at 121oC for 15 minutes

71

APPENDIX K

Preparation for lipase activity


Yeast extract 5.0

Bacto-Tryptone 10.0

Methylene blue 0.15

Agar 20.0

Tween 80 1% (v/v)

Method:

1. All the chemical composition above was dissolved in 1000 mL of distilled

water and the pH was adjusted to 6.8.

2. The medium was then autoclaved at 121oC for 15 minutes

screening and characterization of pha producing bacteria from activated sludge

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