© FITRI BUDIYANTO 2017 iii Dedication To my beloved family iv ACKNOWLEDGMENTS In the name of Allah, the most gracious, most compassionate, most merciful and all the praises and thanks be to Allah that has given me the opportunity and capability to finish my study in KFUPM. I am deeply indebted and most sincere appreciation goes to my advisor, Dr. Assad Al- Thukair, for knowledge, patience, encouragements, and guidance he has shown me over the past few years that I have been with this department, be it scientifically, personally, or academically. I would like to thank my thesis committee members for their support and sharing their knowledge over the years: Dr. Alexis Nzila and Dr. Musa Mohammed Musa. My thanks also are addressed to my wonderful family: my parents, for their endless pray, love and support; my brother and sisters, for keeping my spirit and encouragement during my master journey; and lastly — to my wife for your endless love, support and encouragement has been most influential and inspiring. v TABLE OF CONTENTS ACKNOWLEDGMENTS .............................................................................................. V TABLE OF CONTENTS .............................................................................................. VI LIST OF TABLES ......................................................................................................... X LIST OF FIGURES ...................................................................................................... XI LIST OF ABBREVIATION ....................................................................................... XVI ABSTRACT ............................................................................................................. XVII XIX ................................................................................................................. ملخص الرسالة CHAPTER 1 INTRODUCTION......................................................................................1 1.1 Background overview ............................................................................................... 1 1.2 Problem statement .................................................................................................... 3 1.3 Objectives ................................................................................................................. 4 CHAPTER 2 LITERATURE REVIEW ...........................................................................6 2.1 Arabian Gulf: A potential habitat to discover halophilic bacteria degrading organic compound .................................................................................... 6 2.2 Produced water: One of hypersaline source containing organic pollutants ................. 8 2.3 Pyrene: A persistent Polycyclic Aromatic Hydrocarbon ............................................ 9 2.4 Halophilic Bacteria ................................................................................................. 12 vi 2.4.1 Phylogenic tree of halophilic bacteria .................................................................... 12 2.4.2 Morphology and Physiology of Halophiles ........................................................... 14 2.4.3 The Novel Industrial Application of Halophiles .................................................... 15 2.5 Biodegradation of pyrene ........................................................................................ 18 CHAPTER 3 MATERIAL AND METHODS ................................................................ 24 3.1 The Origin of sample .............................................................................................. 25 3.2 Preparation of the media ......................................................................................... 25 3.3 Enrichment and isolation of bacteria ....................................................................... 26 3.4 Morphological characterization of isolates .............................................................. 26 3.4.1 Morphological characterization of isolates by light microscopy ...................... 26 3.4.2 Morphological characterization of isolates by Electron Microscopy ............... 27 3.5 Molecular identification of isolates ......................................................................... 27 3.6 Phylogenic analysis of bacteria isolates ................................................................... 28 3.7 The observation of strains growth under the presence of pyrene .............................. 28 3.8 Quantification of pyrene biodegradation by halophilic bacteria ............................... 29 3.9 Data analysis .......................................................................................................... 31 CHAPTER 4 RESULTS ................................................................................................ 32 4.1 Isolation of consortia by enrichment ....................................................................... 32 4.2 The isolation of individual strains or colonies ......................................................... 32 4.3 Colony Characterization ......................................................................................... 33 vii 4.4 Species identification by molecular techniques ....................................................... 37 4.4.1 Bacteria preparation ....................................................................................... 37 4.4.2 16s rRNA sequence and species identification ................................................ 37 4.4.3 Phylogeny tree ............................................................................................... 38 4.5 The growth of isolates under different environmental conditions ............................ 41 4.5.1 Standard conditions of growth ........................................................................ 41 4.5.2 Investigation of the optimum conditions of growth ......................................... 43 4.5.3 Effect of initial pyrene concentration .............................................................. 55 4.6 The quantification of pyrene during incubation periods ........................................... 59 4.6.1 Standard curve and the mass spectrum of pyrene ............................................ 59 4.6.2 The degradation of pyrene by Idiomarina sp. 10PY1A ................................... 62 4.6.3 The degradation of pyrene by Halomonas shengliensis 10PY2B .................... 63 4.6.4 The degradation of pyrene by Halomonas Smyrnensis 20PY1A ..................... 64 4.7 The capability of isolates to grow in the presence of different types of carbon source .......................................................................................................... 65 4.7.1 The growth of Idiomarina sp. 10PY1A in the presence of different type of carbon sources ................................................................................... 65 4.7.2 The growth of Halomonas shengliensis 10PY2B under different types of carbon source ................................................................................... 67 4.7.3 The growth of Halomonas smyrnensis 20PY1A under different types of carbon sources .................................................................................. 68 CHAPTER 5 DISCUSSION .......................................................................................... 71 5.1 Isolate characterization: Morphology and Molecular technology ............................. 71 5.2 Isolates growth at various conditions in the presence of pyrene as a sole carbon source .......................................................................................................... 74 5.2.1 The effect of pH on the growth of isolated strains ........................................... 74 5.2.2 The effect of temperature on the growth of isolated strains ............................. 76 viii 5.2.3 The effect of salinity on the growth of the isolates .......................................... 77 5.2.4 The effect of initial pyrene concentration on the growth of isolates ................ 78 5.3 The degradation rate of pyrene by isolated strains ................................................... 79 5.4 The growth of isolated strain in the presence of other aromatic compounds as a sole carbon source ......................................................................... 80 CHAPTER 6 CONCLUSION AND RECOMMENDATION......................................... 82 6.1 CONCLUSION ...................................................................................................... 82 6.2 RECOMMENDATION .......................................................................................... 83 REFERENCES .............................................................................................................. 84 VITAE ........................................................................................................................ 102 ix LIST OF TABLES Table 1. The physical character, carcinogenic potency and toxicity rank of 16 PAHs as USEPA’s list for organic hazardous pollutant ................................... 11 Table 2. The application of halophiles in many sectors of industry ................................. 16 Table 3. The application of microbes either halophiles or non-halophiles in degrading PAHs ............................................................................................... 19 Table 4. The morphological observation of isolated strains ............................................ 33