production of surfactin by using local isolates of

PRODUCTION OF SURFACTIN BY USING LOCAL ISOLATES OF Bacillus subtilis

AMENA A. ABDULRAZEG

(Matric No. 3090096)

UNIVERSITI SAINS ISLAM MALAYSIA


AMENA A. ABDULRAZEG (Matric No. 3090096)

Thesis Submitted in Fulfillment of the Requirement for the Degree of MASTER OF SCIENCE AND TECHNOLOGY

Faculty of Science and Technology UNIVERSITY SAINS ISLAM MALAYSIA

NILAI

June 2012

i

AUTHOR DECLARATION

بسم اهللا الرحمن الرحيم

I hereby declare that the work in this thesis is my own except for quotations and

summaries which have been duly acknowledged.

Date: 13th June, 2012 Signature:

Name: Amena A. Abdulrazeg Address: P13-B-10-08 Sri Cempaka, Kajang 43000, Selangor

ii

DEDICATION

To

My parents for the kindness, love and support:

Ali Abdulrazeg AlAker

and

Mabrouka Mohammed ALGala

My brothers for advice, support, inspiration and willingness to share bright thoughts

with me:

Dr. Aied Ali Abdulrazeg

and

Dr. Ala Ali Abdulrazeg

iii

BIODATA OF AUTHOR

The author was born in Al Beda city, Libya on 14th December 1986. She received the

Bachelor degree in Science -Chemistry department- from Omar Al Muktar University

of Al Beda, Libya, in 2008. The author is doing her Master in Science and

Technology, Universiti Sains Islam Malaysia (USIM). Her research interest is in the

production of surfactin by various strains of isolates of Bacillus subtilis.

iv

APPROVAL

This thesis, entitled "Production of Surfactin by Using Local Isolates of Bacillus subtilis” submitted to the Faculty of Science and Technology (FST), Universiti Sains Islam Malaysia (USIM) and was accepted as fulfillment of the requirements for the degree of Master of Science.

Supervisor’s signature

DR. MOHD HAFEZ BIN MOHD ISA, PH.D, Faculty of Science and Technology, Universiti Sains Islam Malaysia

Date: 13th June, 2012

v

ACKNOWLEDGEMENTS

In the name of Allah, The Most Graceful, The Most Merciful.

All praise is for Allah S.W.T, Lord of the Universe, for giving me the strength in the journey to complete this thesis.

First and foremost, I would like to express my deepest gratitude and appreciation to

my supervisor, Dr. Mohd Hafez Bin Mohd Isa, for his great concern, advices,

patience, persistant encouragement and invaluable assistance from the beginning till the

end of this study, I consider myself very lucky to be given this honour to work with him.

I would like also to convey my thankfulness to Ms. Normah Haron for the HPLC

instrumentation guidance and support, for giving immortal collaboration and support.

To Mr. Mohd Rizal, my co-supervisor, Dr. Siti Salhah Binti Othman, I would like to

address a greatest gratitude to both of you for every unlimited guidance that you gave

to me all this time.

Next, I would like to thank my parents for all your love, guidance and support

always. I treasure you more than you could possibly know. You truly are the best

parents anyone could wish for. Also I would like to thank to my brothers

“Mohammed, Salem & Ahmed”, my sisters “Nada & Abeer” and my sisters in low

“Gada & Asma” for their support and encouragement. Without them, life is

meaningless and efforts are vacant.

Lastly, I would like to express my sincere gratitude to University Sains Islam

Malaysia, lecturers and staff those who had been involved in contributing their time,

effort and support me in making this project a successful reality. I am most fortunate

to have the advice and guidance of many talented people, whose knowledge have

enhanced this project in so many ways.

vi

ABSTRAK

PENGHASILAN SURFAKTIN DENGAN MENGGUNAKAN PENCILAN TEMPATAN DARI Bacillus subtilis

Bacillus subtilis diketahui berupaya mensintesis surfaktin dengan ciri tindakan aktif permukaan yang menakjubkan dan mempamerkan pelbagai aktiviti biologikal. Memandangkan pencilan tempatan B. subtilis tempatan adalah banyak dan berkos rendah, pencilan tempatan yang dinamai sebagai 1M, 3M, 7M, dan 8M telah dikaji melalui fermentasi kelalang goncang dan kromatografi cecair berprestasi tinggi (HPLC). Kerja-kerja eksperimen telah dijalankan untuk mengubahsuai kaedah kuantifikasi dan identifikasi surfaktin melalui teknik HPLC, serta menilai keupayaan empat pencilan tempatan dalam penghasilan surfaktin. Proses fermentasi telah dijalankan dengan menggunakan formulasi media Cooper dengan keadaan 30 ⁰C, 200 pusingan dalam seminit selama 168 jam. Surfaktin yang terhasil dibandingkan dengan pencilan komersial B. subtilis ATCC 21332. Tambahan lagi, kesan kepekatan yang berbeza oleh glukosa dan ion mangan (II), Mn2+ telah dikaji ke atas penghasilan surfaktin. Keputusan kajian mendapati hasil surfaktin dan tumbesaran bakteria yang maksimum dicapai oleh pencilan tempatan sewaktu tempoh inkubasi 96 jam manakala bagi pencilan komersial pada 72 jam. Pencilan 3M menghasilkan jumlah penghasilan surfaktin tertinggi manakala jumlah yang terendah telah dihitung daripada pencilan 1M. Penghasilan surfaktin oleh B. subtilis ATCC 21332 didapati lebih tinggi secara signifikan pada (P≤ 0.01) berbanding pencilan 1M, bagaimanapun tiada perbezaan yang signifikan pada jumlah penghasilan surfaktin di antara pencilan ATCC 21332 dan 3M. Tambahan 0.5 mM MnSO4 dan 40 mg/L glukosa ke dalam media fermentasi mampu mempertingkatkan penghasilan surfaktin. Penapisan dan pemekatan pati fermentasi telah disempurnakan dengan menggunakan alat pengempar ultrafiltrasi (UF) sebelum pencirian jisim molekul melalui kromatografi cecair-spektrometri jisim (LC-MS). Siri isoform surfaktin yang dihasilkan oleh pencilan 1M dan 3M mempunyai persamaan dengan surfaktin piawai yang juga dihasilkan oleh B. subtilis ATCC 21332.

vii

ABSTRACT


Bacillus subtilis is able to synthesize surfactin with excellent surface-active properties and biological activities. Since local isolates of B. subtilis are abundant and cheap, quantification and identification of surfactin produce by four local isolates of B. subtilis named as 1M, 3M, 7M, and 8M were studied by through shake flasks fermentation and high performance liquid chromatography (HPLC). The experimental work was conducted to develop modified method of HPLC technique for surfactin quantification and identification, as well as to assess the ability of four local isolates to produce surfactin using Cooper’s media in shake flasks fermentation under the condition of 200 rpm for 168 h at 30oC. The produced surfactin were compared with the commercial strain of B. subtilis ATCC 21332. In addition, various concentrations of glucose and manganese were added to the fermentation media to investigate its effects on surfactin production. Increasing the solvent system of acetonitrile-deionised water at 80:20 % (v/v) with 1.5 mL/min flowrate showed significant rapid elution of surfactin isoform which also proved that four local isolates strains have the ability to produce surfactin. The results obtained show that the maximum surfactin production and bacterial growth of both local and commercial strains were at 96 hours and 72 hours of fermentation, respectively. Moreover, B. subtilis 3M showed the highest amount of surfactin production, while B. subtilis 1M strain produced the lowest amount. For B. subtilis 8M, maximum surfactin production was achieved at around 72 h of fermentation, with surfactin yield of 105 ± 12 mg/L and the production of surfactin for B. subtilis 7M in 96 h of fermentation, with surfactin yield of 86 ± 11 mg/L. In addition, Surfactin production of B. subtilis ATCC 21332 strain was significantly higher (P≤ 0.01) in comparison to B. subtilis 1M, whereas no significant difference in surfactin production between B. subtilis ATCC 21332 and B. subtilis 3M. Addition of 0.5mM MnSO4 and 40 mg/L of glucose to the fermentation media managed to increase the yield of surfactin. Finally, fermentation broths recovered and concentrated with Ultrafiltration (UF) device were analyzed with liquid chromatography-mass spectrometer (LC-MS) to determine the molecular mass of surfactin isoforms. The isolates produced series of surfactin isoforms except for B. subtilis 7M. The isolates of B. subtilis 1M, B. subtilis 3M, and B. subtilis 8M produced series of surfactin isoform, which have similarity with surfactin standard isoforms. Furthermore, the local isolates produced new surfactin isoform which has molecular mass of 927 Da. B. subtilis ATCC 21332 as well produced a new surfactin isoform with molecular mass of 1101 Da.

viii

ملخص

ستخدام العزالت محلية من إب Surfactin نتاج إ العصوية الرقيقة بكتيريا

ةخاصيب متازت تيال (Surfactin) مادة قادر على تخليق Bacillus subtilis)( البكتيريا العصوية الرقيقةبما إن العزالت المحلية . نشاطات بيولوجية متعددةامتالآها يلإ األضافهبذات آفائة عالية لتوتر السطحياأستخدام أربعة عزالت محلية من ات تم زالممي ةومن خالل هذمتوفره ورخيصة الثمن لبكتريا العصوية الرقيقة ل

وتم دراستها عن طريق 8M) و 1M،3M ، 7M(وتندرج تحت أالسماء آالتيه البكتريا العصوية الرقيقة بإستخدام جهاز آروماتوجرافيا السائل ذي الكفاءة العاليةيضا أو shake flask)( تخميرها في القوارير هزالتجريبي لتطوير أسلوب تعديل ةوتمت االجراءات المعملي. (Surfactin) المسماه لتحديد آمية الماده المنتجة

لتقييم أيضاو ،(Surfactin)نتاج إ ةتقنية جهاز آروماتوجرافيا السائل ذي الكفاءة العالية للتحديد نوع وآمييسمي مخمر بتخمير العزالت المحلية في وسط (Surfactin)إنتاج علي ةمدي قدرة العزالت المحلية األربع

(Cooper’s media) ساعة وعند 168 لمدة ،لفة في الدقيقة 200تحت الضروف التالية القوارير هز فيالمنتج من الكمية . )Incubator shake(ة ويتم تحضينها في جهاز يسمي مئوي 30درجة حرارة

(Surfactin) والتي تسمي التجارية العصوية الرقيقة البكتيرياقارن مع تعنئذ(B. subtilis ATCC أضيفت إلى وسط لدراسة مدي قد ترآيزات مختلفة من الجلوآوز والمنغنيز الباإلضافة إلى ذلك، . 21332)

-acetonitrileزيادة نظام المذيبات ن إومن النتائج المتحصل عليها لوحظ .(Surfactin)نتاج إعلى هاثيرأتdeionised water 80:20عند % (v/v) فضل نتيجة أاظهرت قد مل في الدقيقه 1.5السريان معدلمعالمحلية األربع لديها أيضا أن سالالت ناأثبت من خاللها التيو) Surfactin isoform (لعملية شطف وإنتاج

النتائج التي تم الحصول عليها تشير إلى أن الحد األقصى للنمو الجرثومي . Surfactin)(القدرة على إنتاج ساعة من 72ساعة و 96 خاللمن السالالت المحلية والتجارية على حد سواء آانت (surfactin)نتاج أو

في انتاج أعلى آمية )3M) B. subtilisالبكتيريا المحلية وعالوة على ذلك، نجحت. التخمير، على التوالي. Surfactin)(نتجت أدني آمية من أ B. subtilis) (1Mالبكتيريا المحلية في حين أن Surfactin)(من

≥P(آان أعلى بكثير (B. subtilis ATCC 21332)التجارية ةبواسطة السالل Surfactin)(إنتاج 0.01 ( .B)بين Surfactin)(، بينما ال يوجد فرق آبير في إنتاج B. subtilis) (1M البكتيريا المحلية بالمقارنة مع

subtilis ATCC 21332) و(B. subtilis 3M) .جرام في اللتر 40من المنعنيز و ليمولم 0.5إضافة بيتم معالجة و ،خيراأو. Surfactin)(زيادة نسبة إنتاج هامن الجلوآوز الي الوسط المخمر تمكنا من خالل

لوبعد ذلك يتم تحديد الكتلة الجزيئية جهاز الترشيح الفائق ترآيز الوسط المتخمر عن طريق استخدام )(surfactin isoform العزالت . جهاز مطياف الكتلةبإستخدام(B. subtilis 1M) و(B. subtilis 3M)

Surfactin standard(المنتجه من سلسلة وبمقارنتها مع )Surfactin isoform(من ةأنتجة سلسلقد isoforms( الساللة التجاريه بواسطةالمنتج عن (B. subtilis ATCC 21332) لحظ ان هناك تشابه بينهما

.ةالمحليالبكتيريا وبذلك يمكن تحديد الكتلة الجزيئيه للسالالت

ix

TABLE OF CONTENT

page

AUTHOR DECLARATION i

DEDICATION ii

BIODATA OF AUTHOR iii

ACKNOWLEDMENTS iv

ABSTRAK v

ABSTRACT vi

vii ملخص

TABLE OF CONTENT viii

LIST OF TABLES x

LIST OF FIGURES xi

LIST OF APPENDICES xiii

ABBREVIATIONS xiv

CHAPTER I: INTRODUCTION 1

CHAPTER II: LITERATURE REVIEW

2 .1 Surfactant 4

2.2 Biosurfactants 5

2.3 Surfactin 7

2.4 Biological Properties of Surfactin 8

2.5 Structure and physicochemical properties 9

2.6 Isoforms of Surfactin 12

2.7 Surfactin Biosynthesis 13

x

2.8 Potential Applications of Surfactin 15

2.8.1 Anti-Bacterial Activity 15

2.8.2 Anti-Viral Activity 15

2.8.3 Antitumor Activity 16

2.8.4 Anti-Mycoplasma Effect 16

2.8.5 Toxicity 16

2.9 Recent Trends in Surfactin Production 17

2.10 Local Isolates of Bacillus subtilis 18

CHAPTER III: DEVELOPMENT OF HIGH PERFORMANCE

LIQUID CHROMATOGRAPHY (HPLC) METHOD

3.1 Introduction 19

3.2 Materials and Methods 20

3.2.1 Preparation of Surfactin Standard Solution 20

3.2.2 High Performance Liquid Chromatography

System for Surfactin standard Analysis 21

3.3 Results and Discussion 22

3.3.1 Effect of Flowrate 22

3.3.2 Effect of Acetonitrile Concentration 24

3.4 Chromatographic Characterization 26

3.5 Surfactin Standard Calibration Curve 28

3.6 Conclusion 29

CHAPTER IV: PRELIMINARY STUDY OF SURFACTIN

PRODUCTION

4.1 Introduction 30

4.2 Materials and Methods 32

4.2.1 Fermentation 32

4.2.2 Determination of Bacterial Growth 32

4.2.3 Quantitative Analysis of Surfactin 32

xi

4.2.4. Liquid Chromatography-Mass Spectrometer

(LC-MS) Analysis 33

4.2.5 Statistical Analyses 33


4.3.1 Time Course of Surfactin Production and

Cell Growth 34

4.3.2 Chromatographic Characterization 37

4.3.3 Liquid Chromatography-Mass Spectrometry

Analysis (LC-MS) 39

4.3.3.1 Analysis of Standard Surfactin by LC-MS 41

4.3.3.2 Surfactin isoforms of B. subtilis 1M Isolate 42




4.3.3.6 Surfactin isoforms of B. subtilis ATCC 21332 45

4.4 Conclusion 46

CHAPTER V: ENHANCEMENT OF SURFACTIN PRODUCTION

5.1 Introduction 47

5.2 Production of surfactin by Bacillus subtilis 48

5.2.1 Microorganism 48

5.2.2 Culture medium 48

5.2.3 Inoculum and culture conditions 48

5.2.4 Effect of Glucose (C6H12O6) Concentration as

a Carbon Source 49

5.2.5 Effect of Manganese Sulfate (MnSO4)

Concentration 49

5.3 Analytical measurements 49

5.3.1 Measurement of Bacterial Growth 49

5.3.2 Surfactin Concentration Analysis 50


xii

5.4.1 Effect of Glucose (C6H12O6) Concentration

As a Carbon Source 50

5.4.2 Effect of Manganese Sulfate (MnSO4)

Concentration 52

5.5 Conclusion 56

CHAPTER VI: SUMMARY 57

REFERENCES 59

APPENDICES 70

xiii

LIST OF TABLES

Page

Table 1: Preparation of surfactin standard for HPLC analysis 21

Table 2: Surfactin standard isoforms 41

Table 3: Surfactin isoforms produced by B. subtilis 1M 42



Table 6: Surfactin isoforms produced by B. subtilis ATCC 21332 44

xiv

LIST OF FIGURES

Page

Figure 1: Primary structure of surfactin 9

Figure 2: Three-dimensional structure of surfactin peptide moiety 10

Figure 3: Schematic representation of the surfactin synthetase 14

Figure 4: High Performance Liquid Chromatography 21

Figure 5: Effect of flowrate tested at 1.5 mL/min 23




Figure 9: Effect of acetonitrile-deionised water at 85:15 % (v/v),

with the flowrate tested at 1.0 mL/min 25







Figure 13: a) Chromatogram of surfactin standard analyzed by Abdel-

Mawgoud et al., (2009); b) chromatogram of surfactin

standard (500 mg/L) in methanol 27

Figure 14: Calibration curve of surfactin standard 29

xv

Figure 15: Gram positive Bacillus ubtilis bacteria on nutrient agar (NA) 30

Figure 16: Time course of B. subtilis 3M growth (open symbols) and, surfactin concentration (Solid symbols) 34




Figure 20: Time course of B. subtilis ATCC21332 growth (open symbols) and, surfactin concentration (Solid symbols) 37

Figure 21: HPLC chromatogram of surfactin standard from Sigma 39

Figure 22: HPLC chromatogram of B. subtilis 3M isolate 39



Figure 25: HPLC chromatogram of B. subtilis ATCC21332 40


Figure 23: Effects of glucose concentration on bacteria growth of Bacillus 51

Figure 24: Effects of glucose concentration on surfactin production 52

Figure 25: Effects of MnSO4 concentration on bacteria growth of Bacillus 53

Figure 26: Effects of MnSO4 concentration on surfactin production 53

Figure 27: HPLC chromatogram of surfactin standard from Sigma 54


xvi



Figure 31: HPLC chromatogram of B. subtilis ATCC21332 56


xvii

LIST OF APPENDICES

Page

Appendix A

Figures A: Effect of flowrate tested at 1.5 mL/min on HPLC spectrograms

of surfactin standard from Sigma 70

Figure 1A: HPLC spectrogram for 10 mg/L of surfactin standard from Sigma 70




Appendix B

Figures B: Effect of flowrate tested at 0.5 mL/min on HPLC spectrograms


Figure 1B: HPLC spectrogram for 10 mg/L of surfactin standard from Sigma 71




Appendix C

Figures C: Effect of flowrate tested at 2.0 mL/min on HPLC spectrograms


Figure 1C: HPLC spectrogram for 10 mg/L of surfactin standard from Sigma 72




xviii

Appendix D

Figures D: Effect of flowrate tested at 1.0 mL/min on HPLC spectrograms


Figure 1D: HPLC spectrogram for 10 mg/L of surfactin standard from Sigma 73




Appendix E

Figures E: Effect of acetonitrile-deionised water at 85:15 % (v/v), with

1.0 mL/min on HPLC spectrograms of surfactin standard from

Sigma 74

Figure 1E: HPLC spectrogram for 10 mg/L of surfactin standard from Sigma 74




Appendix F

Figures F: Effect of acetonitrile-deionised water at 85:15 % (v/v), with


Sigma 75

Figure 1F: HPLC spectrogram for 10 mg/L of surfactin standard from Sigma 75




xix

Appendix G

Figures G: Effect of acetonitrile-deionised water at 85:15 % (v/v), with


Sigma 76

Figure 1G: HPLC spectrogram for 10 mg/L of surfactin standard from Sigma 76




Appendix H

Figures H: Effect of acetonitrile-deionised water at 85:15 % (v/v), with


Sigma 77

Figure 1H: HPLC spectrogram for 10 mg/L of surfactin standard from Sigma 77




Appendix K

Figures K: MS chromatogram results for all strains 78

Figure 1K: Chromatogram analysis done by LCMS for surfactin standard 78

Figure 2K: Chromatogram analysis done by LCMS for surfactin produced

by Bacillus 1M 79


by Bacillus 3M 80


by Bacillus 7M 81

xx


by Bacillus 8M 82


by B. subtilis ATCC 21332 83

Appendix I

Data I: The result for statistical analyses for the B. subtilis Isolates 84

Appendix I

Presentation and Demonstration 86

xxi

ABBREVIATIONS

mN/m Millinewnon Per Meter

NH4NO3 Ammonium nitrate

W/V weight/volume

ATCC American Type Culture Collection

M.W Molecular Weight

NMR Nuclear magnetic resonance

CMC Critical micelle concentration

HPLC High performance liquid chromatography

TE Thioesterases

cAMP Cyclic adenosine monophoshate

TLC Thin-layer chromatography

B. subtilis Bacillus subtilis

ACN Acetonitrile

TFA Trifluoroacetic acid

VWD Variable wavelength detector

rpm Revolutions per minute

TPA Total peak area

PLA2 Phospholipase A2

KH2PO4 Potassium phosphate

Na2HPO4 Sodium phosphate

CaCL2 Calcium chloride

MgSO4 Magnesium sulfate

xxii

MnSO4 Manganese Sulfate

FeSO4 Ferrous sulfate

EDTA Na2 salt of Ethylenedamien tetra acetic acid

NA Nutrient agar

OD600nm Optical density at 600 nm

LC-MS Liquid Chromatography-Mass Spectrometer

Min Minutes

H Hours

LC-MS Liquid Chromatography-Mass Spectrometer

°C Degrees Celsius

Da Dalton

MWCO Molecular weight cut off

59

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