universiti putra malaysiapsasir.upm.edu.my/id/eprint/39201/1/fbsb 2012 33r.pdf · universiti putra...

UNIVERSITI PUTRA MALAYSIA

MAZURIN BINTI MAHAMOOD

FBSB 2012 33

BIODEGRADATION OF SODIUM DODECYL SULPHATE USING LOCALLY ISOLATED Pseudomonas aeruginosa sp. STRAIN D1

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BIODEGRADATION OF SODIUM DODECYL

SULPHATE USING LOCALLY ISOLATED

Pseudomonas aeruginosa sp. STRAIN D1


MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2012

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BIODEGRADATION OF SODIUM DODECYL SULPHATE

USING LOCALLY ISOLATED Pseudomonas aeruginosa sp. STRAIN D1

By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the

Requirements for the Degree of Master of Science

February 2012

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in

fulfillment in fulfillment of the requirements for the degree of Master of Science

BIODEGRADATION OF SODIUM DODECYL SULPHATE

USING LOCALLY ISOLATED Pseudomonas aeruginosa sp. STRAIN D1

By


February 2012

Chairman : Professor Mohd Arif Syed, PhD

Faculty : Biotechnology and Biomolecular Sciences

Surfactants are synthetic organic chemicals that are formulated to have

cleansing or solubilisation properties. With the development of the industrial

economy and increase in population density, surfactants have become one of the

most widely disseminated toxic substances to enter the aquatic environment,

creating a serious environmental problem. High concentration of SDS in the

environment may give negative influences to the folding of a polypeptide chain

and changes the surface charge of the molecule in the organisms and thus will

disrupt the ecosystem. Their toxicities to organisms have been demonstrated

previously by many reserchers (Ying, 2006; Singh et al., 2002; Lewis, 1991;

Utsunomiya et al., 1997; Mori et al., 2002). Therefore, biodegradation of SDS

is important in order to ensure low concentration of SDS in the environment.

Local microorganisms were used in this study as it has high ability to adapt with

the local environment such as temperature, humidity and so on. The main

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objective of this study was to isolate, characterize and finally immobilize a local

bacterium with the potential to degrade Sodium Dodecyl Sulphate (SDS), a

widely used anionic surfactant. Samples for this study were collected from

detergent-contaminated area from several locations in Malaysia including from

car wash and laundry’s outlets, drains, sludge and soil samples as mentioned in

methodology (section 3.2). Screening was carried out by the conventional

enrichment culture technique and the bacterium was tentatively identified as

Pseudomonas aeruginosa sp. strain D1 HM852751 using BiologTM GN plates

and partial 16S rRNA phylogeny. The optimal growth conditions in minimal

medium and for degradation of SDS by Pseudomonas aeruginosa sp. strain D1

HM852751 were at 30°C and at pH 6.5 using phosphate buffer system. Sodium

nitrate; at 8 gL-1

was found to be the best nitrogen source. The isolated strain

exhibited optimum growth at SDS concentration of 1 gL-1

but can tolerate up to

14 gL-1

SDS, indicating that this isolate was able to survive in a relatively high

concentration of SDS. 100% of 1 gL-1

SDS was completely degraded after 5 and

2 days of incubation before and after optimization, respectively. Encapsulation

or immobilization of microorganisms of interest is a new technique and has

proven to be more efficient in biodegradation of pollutants. Hence the

Pseudomonas aeruginosa sp. strain D1 HM852751 was immobilized using

gellan gum to enhance the degradation of SDS by the selected isolate.

Optimizations of different immobilization parameters were carried out. The

optimum gellan gum concentration for immobilized Pseudomonas aeruginosa

sp. to degrade SDS ranged from 0.8% to 0.85%. The optimum cell density was

between 40 gL-1

to 50 gL-1

and the optimum bead size was 4.5 mm with the

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initial cell loading of 250 beads. 1 gL-1

of SDS was successfully degraded

within 8 hours by immobilized cells compared to 20 hours by the freely

suspended cells which was a substantial reduction in the degradation time. The

immobilized cells can be used up to 20 cycles with approximately 100%

reduction of SDS. These findings indicates Pseudomonas aeruginosa sp. strain

D1 have high ability to degrade SDS when it was immobilized in gellan gum

and has high potential for future research.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia

sebagai memenuhi keperluan untuk ijazah Master Sains

BIOPENGURAIAN SODIUM DODECYL SULPHATE

MENGGUNAKAN Pseudomonas aeruginosa sp. STRAIN D1

TEMPATAN.

Oleh


Februari 2012

Pengerusi : Profesor Mohd Arif Syed, PhD

Fakulti : Bioteknologi dan Sains Biomolekul

Surfaktan adalah bahan kimia organik sintetik yang diformulasi untuk

mempunyai sifat pelarut. Dengan peningkatan ekonomi perindustrian dan

peningkatan populasi, surfaktan menjadi salah satu bahan toksik yang tersebar

secara meluas yang masuk ke persekitaran akuatik menyebabkan masalah

pencemaran persekitaran yang serius. Kepekatan SDS yang tinggi di persekitaran

akan memberi kesan negatif pada ikatan rantai polipeptida dan mengubah

permukaan cas pada molekul dalam organisma and seterusnya akan

mengganggu ekosistem. Kesan toksik kepada organisma telah dilaporkan

sebelum ini oleh ramai penyelidik. (Ying, 2006; Singh et al., 2002; Lewis,

1991; Utsunomiya et al., 1997; Mori et al., 2002). Oleh itu, biodegradasi SDS

adalah penting untuk memastikan kepekatan SDS adalah rendah di persekitaran.

Mikroorganisma tempatan digunakan memandangkan ia mempunyai kebolehan

yang tinggi untuk menyesuaikan diri dengan persekitaran tempatan seperti suhu,

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kelembapan dan lain-lain. Objektif utama kajian ini adalah untuk memencilkan

dan mencirikan bakteria tempatan dan seterusnya menyekat-gerak bakteria yang

mempunyai potensi untuk menguraikan Sodium Dodecyl Sulphate (SDS), iaitu

satu surfaktan anionik yang digunakan secara meluas. Sampel untuk kajian ini

diambil daripada kawasan yang tercemar dengan bahan pencuci dari beberapa

lokasi di Malaysia termasuk dari tempat membasuh kereta dan kedai dobi,

longkang, lumpur dan sampel tanah seperti yang dinyatakan di dalam

metodologi (seksyen 3.2). Pemilihan dijalankan menggunakan teknik

pengayaan kultur konvensional dan bakteria tersebut dikenali sebagai

Pseudomonas aeruginosa sp. strain D1 HM852751 menggunakan plate

BiologTM GN dan filogeni separa 16S rRNA. Keadaan optimum bagi

pertumbuhan dan degradasi SDS dalam media minimal oleh Pseudomonas

aeruginosa sp. strain D1 HM852751 adalah pada 30°C dan pH 6.5

menggunakan sistem penimbal fosfat. Natrium Nitrat pada 8 gL-1

merupakan

sumber nitrogen yang terbaik. Bakteria ini menunjukkan pertumbuhan optimum

pada kepekatan 1 gL-1

SDS tetapi masih boleh bertahan sehingga 14 gL-1

SDS,

menunjukkan bakteria ini berkemampuan untuk hidup dalam kepekatan SDS

yang tinggi. Degradasi 1 gL-1

SDS sebanyak 100% berlaku selepas inkubasi

selepas 5 dan 2 hari pada sebelum dan selepas proses pengoptimuman. Kaedah

sekat-gerak mikroorganisma terpilih merupakan teknik yang baru dalam

membiodegradasi bahan tercemar dan terbukti lebih efisien dalam biodegradasi

bahan-bahan tercemar. Oleh itu, Pseudomonas aeruginosa sp. strain D1

HM852751 disekat-gerak menggunakan gellan gum untuk meningkatkan

degradasi SDS oleh isolat yang telah dipilih. Pengoptimuman parameter untuk

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prosedur sekat-gerak dilakukan. Kepekatan gellan gum optimum untuk

Pseudomonas aeruginosa sp. yang disekat-gerak untuk degradasi SDS adalah di

antara 0.8% to 0.85%. Ketumpatan sel optimum adalah di antara 40g L-1

to 50g

L-1

dan saiz butir manik optimum adalah 4.5mm dengan permulaan bilangan sel

pada 250 butir manik. 1 gL-1

SDS telah berjaya diuraikan dalam masa 8 jam

oleh sel yang disekat-gerak berbanding 20 jam oleh sel bebas. Sel yang disekat-

gerak ini juga boleh digunakan sehingga 20 kitaran dengan penurunan hampir

100% SDS. Penemuan ini menunjukkan Pseudomonas aeruginosa sp. Strain D1

mempunyai keupayaan yang tinggi dalam degradasi SDS apabila ia disekat

gerak menggunakan gellan gum dan ia mempunyai potensi yang tinggi untuk

penyelidikan di masa hadapan.

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ACKNOWLEDGEMENTS

All praise due to Almighty Allah, the most Merciful and most Benevolent. The

completion of this study would not be possible had it not been for His will and

favor. I wish to extend my deepest appreciation to both my supervisors Prof. Dr.

Mohd. Arif Syed and Associate Prof. Dr. Mohd Yunus Abd. Shukor for their

advices, comments, guidance, inspiration, supervision and encouragement.

I would like to thank School of Graduate Studies (SGS), Universiti Putra

Malaysia for rewarding me GRF scholarship. To the Department of

Biochemistry, Universiti Putra Malaysia for this opportunity to expand my

knowledge and skills. To the Bioremediation Lab, Department of Biochemistry,

Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia

for the lab equipments and facilities.

My sincere gratitude and appreciation to all members of Bioremediation Lab

and the Laboratory Assistants: En. Hussein and Puan Sharipah for providing

their assistance. Lots of thanks and gratitude to all my colleagues and also to

those who have contribute directly or indirectly in sharing their knowledge,

skills and assistance throughout the course of my study. You all have always

been there for the friendship and encouragement during the most crucial

moments.

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Last but not least, my deepest gratitude and thanks to my beloved parents,

Mahamood Bin Paksu and Habsah Binti Abdullah and my dearest husband

Mohd Hafizul Bin Mohd Jaaffar for his love, understanding, perseverance and

constant prayers. Thanks for encouragement, patience and understanding, this

had helped me to complete this research study. Thank you so much.

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APPROVAL SHEET 1

I certify that a Thesis Examination Committee has met on 13 February 2012 to

conduct the final examination of Mazurin Binti Mahamood on her Master of

Science thesis entitled Biodegradation of SDS By Locally Isolated

Pseudomonas aeruginosa sp. strain D1 in accordance with the Universities

and Universiti College Act 1971 and the Constitution of the Universiti Putra

Malaysia [P.U.(A) 106] 15 March 1998. The committee recommends that the

student be awarded the Master of Science.

Members of the Examination Committee were as follows:

Prof. Madya Dr. Foo Hooi Ling, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Y. Bhg. Prof Dr. Mohd Ali Hassan, PhD

Professor



(Internal Examiner)

Dr. Wan Zuhainis Binti Saad, PhD



(Internal Examiner)

Prof. Madya Dr. Mushrifah Idrid, PhD

Associate Professor

Faculty of Sciences and Technology

Universiti Kebangsaan Malaysia

(External Examiner)

____________________________

Prof. Dr. Zulkarnain Zainal Prof. Dr. Seow Heng Fong

Professor and Deputy Dean

School of Graduate Studies


Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has

been accepted as fulfillment of the requirement for the degree of Master of

Science. The members of Supervisory Committee were as follows:

Mohd Arif Bin Syed, PhD

Professor



(Chairman)

Mohd Yunus Bin Abd. Shukor, PhD

Associate Professor



(Member)

Nor Aripin Shamaan, PhD

Associate Professor



(Member)

________________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

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DECLARATION

I declare that the thesis is my original works except for quotations and citations

which have been duly acknowledge. I also declare that it has not been

previously, and is not concurrently, submitted for any other degree at Universiti

Putra Malaysia or at any other institution.

_____________________________


Date: 13 February 2012

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK v

ACKNOWLEDGEMENTS viii

APPROVAL x

DECLARATION xii

LIST OF TABLES xvii

LIST OF FIGURES xviii

LIST OF ABBREVIATIONS xxi

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 4

2.1 Surfactant 4

2.1.1 Use of surfactants 4 2.1.2 Characteristics of surfactants 5

2.2 Pollution by surfactants 7

2.3 Effects of surfactants 8

2.3.1 Water 8

2.3.2 Soil 8

2.4 Anionic surfactants 9

2.5 Toxic effects of anionic surfactants 9

2.5.1 Effects on biological activity 9

2.6 Detection of surfactants 13

2.6.1 Methylene Blue Active Substances 14

(MBAS) assay

2.7 Bioremediation 15

2.8 Biodegradation of Anionic surfactants 16

2.9 Immobilization of Pseudomonas aeruginosa sp. 20

by gellan gum

2.9.1 Advantages of Immobilization 20

2.9.2 Gellan gum 21

2.9.3 Gellan gum as immobilization agent 22

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3 MATERIALS AND METHODS

3.1 Chemicals and equipments 24

3.2 Bacterial sampling 25

3.2.1 Isolation of SDS-degrading bacteria 26

3.2.2 Maintenance of cultural stock 27

3.3 Screening for SDS-degrading ability 27

3.3.1 Primary screening 28

3.3.2 Secondary screening 28

3.3.3 Methylene blue active substances 29

(MBAS) assay

3.4 Identification 30

3.4.1 Biochemical test 30

3.4.1.1 Gram staining 30

3.4.1.2 Catalase test 31

3.4.1.3 Oxidase test 32

3.4.2 BiologTM

Identification System 32

3.4.3 16S rDNA analysis 34

3.4.4 Phylogenetic tree analysis 35

3.5 Determination of the optimum conditions for 35

bacterial growth and SDS degradation.

3.5.1 The effects of SDS concentrations 37

3.5.2 The effects of nitrogen sources 37

3.5.3 The effects of nitrogen source 38

concentration

3.5.4 The effects of pH 38

3.5.5 The effects of temperature 39

3.5.6 SDS degradation study 39

3.6 Cell immobilization 40

3.7 Optimization of immobilization protocols 41

3.7.1 The effect of composition of gelling 41

component

3.7.2 The effects of initial cell density 41

3.7.3 The effects of the bead sizes 42

3.7.4 The effect of initial cell loading (ICL) 42

3.8 Comparison of SDS-degrading activities 43

between freely-suspended and encapsulated cell

3.8.1 Degradation of different concentration 43

of SDS

3.8.2 Effects of heavy metals 44

3.9 Reusability of immobilized cell 44

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4 RESULTS AND DISCUSSION

4.1 Isolation of SDS-degrading Bacteria 46

4.2 Screening of SDS-degrading Bacteria 48

4.2.1 Primary Screening of SDS-degrading 48

Bacteria

4.2.2 Secondary Screening of SDS-degrading 49

Bacteria

4.3 Identification of SDS-degrading Bacteria 52

4.3.1 Morphological Observation 53

4.3.2 Biochemical Test 54

4.3.2.1 Gram Identification 54

4.3.2.2 Catalase Test 55

4.3.2.3 Oxidase Test 56

4.3.3 BIOLOGTM

Identification Test 56

4.3.4 16S rRNA Analysis 58

4.3.4.1 Polymerase Chain Reaction 58

(PCR)

4.3.4.2 16S rRNA Gene Sequencing 60

4.3.4.3 Phylogenetic Analysis 61

4.4 Optimization of Isolate D 63

4.4.1 The effects of SDS concentrations 63

4.4.2 Optimization of pH 67

4.4.3 Optimization of Temperature 70

4.4.4 Optimization of Nitrogen Source and 72

Nitrogen Concentration

4.5 SDS Degradation Study before and after 75

optimization

4.6 Cell immobilization 77

4.6.1 Optimization of immobilization protocols 79

4.6.1.1 Effect of gellan gum 79

concentration

4.6.1.2 Effect of initial cell density 81

4.6.1.3 Effect of beads size 83

4.6.1.4 Effect of initial cell loading 85

4.6.1.5 Biodegradation of SDS using 87

optimized gellan gum beads

4.6.2 Comparison of SDS-degrading activities 90

between freely-suspended and

immobilized cell

4.6.2.1 Effects of different 93

Concentrations of SDS

4.6.2.2 Effects of heavy metal 98

4.6.3 Reusability of immobilized Pseudomonas 101

aeruginosa sp. Strain D1 in gellan

gum beads

4.6.4 Cell growth patterns in immobilized 104

beads

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5 CONCLUSION 107

REFERENCES 109

Appendix 1: List of Chemicals 119

Appendix 2: List of Instruments 123

Appendix 3: Standard Curve of SDS 124

Appendix 4: Identification of Isolate D1 using BIOLOGTM

125

GN2 Plate

Appendix 5: Sequences of 16S rRNA Isolate D1 126

BIODATA OF STUDENT 127

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