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
MAZURIN BINTI MAHAMOOD
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
MAZURIN BINTI MAHAMOOD
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
MAZURIN BINTI MAHAMOOD
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
MAZURIN BINTI MAHAMOOD
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
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Dr. Wan Zuhainis Binti Saad, PhD
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(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
Universiti Putra Malaysia
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
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Mohd Yunus Bin Abd. Shukor, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
Nor Aripin Shamaan, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
________________________________
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
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.
_____________________________
MAZURIN BINTI MAHAMOOD
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