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Biodegradation of Polycyclic Aromatic Hydrocarbons in Petroleum Oil Contaminating the Environment

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Biodegradation of Polycyclic Aromatic Hydrocarbons in Petroleum Oil Contaminating the Environment Aim of the Work BIODEGRADATION OF POLYCYCLIC AROMATIC HYDROCARBONS IN PETROLEUM OIL CONTAMINATING THE ENVIRONMENT Presented by Abir Moawad Partila A Thesis Submitted to Faculty of Science In Partial Fulfillment of the Requirements for the Degree of Ph.D. of Science (Microbiology) Botany Department Faculty of Science Cairo University (2013) i Aim of the Work ABSTRACT Student Name: Abir Moawad Partila Girgis Title of the thesis: Biodegradation of Polycyclic Aromatic Hydrocarbon in petroleum oil contaminating the environment Degree: Ph. D. (Microbioliogy) Soil and sludge samples polluted with petroleum waste from Cairo Oil Refining Company Mostorod, El-Qalyubiah, Egypt for more than 41 years were used for isolation of indigenous microbial communities. These communities were grown on seven polycyclic aromatic hydrocarbon compounds .Six isolates (MAM-26, 29, 43, 62, 68, 78) were able to grow on different concentrations of five chosen PAHs. The best degraders bacterial isolates MAM-29 and MAM-62 were identified by 16S-rRNA. As Achromobacterxylosoxidans and Bacillus amyloliqueficiensrespectively.The most promising bacterial strain Bacillus amyloliqueficiens have been exposed to different doses of gamma radiation to improve its qualities. Keywords: Polycyclic- Aromatic- Hydrocarbon – Biodegradation-Pollution. Supervisors: Signature 1- Prof. Dr. Youssry Saleh 2-Prof. Dr. Mervat Aly Abou-State Prof. Dr. Gamal Fahmy Chairman of Botany Department Faculty of Science-Cairo University ii Aim of the Work APROVAL SHEET FOR SUMISSION Thesis Title: Biodegradation of Polycyclic Aromatic Hydrocarbons In Petroleum Oil Contaminating The Environment Name of candidate: Abir Moawad Partila This thesis has been approved for submission by the supervisors: 1- Prof. Dr. Youssry Saleh Signature: 2- Prof. Dr. Mervat Ali Abou State Signature: Prof. Dr. Gamal Fahmy Chairman of Botany Department Faculty of Science- Cairo University iii Aim of the Work TO WHOM IT MAY CONCERN This Thesis has not been previously submitted for any degree at this or at any other university. Signature Abir Moawad Partila iv Aim of the Work v Aim of the Work Acknowledgement I would like to acknowledge with deep gratitude Prof. Dr. Youssry Saleh, Professor of Microbiology, Botany Department, Faculty of Science, Cairo University for valuable advice and his direct supervision and support during the stages of this work. From my deep heart my thanks are for Prof. Dr. Mervat Aly Abou-State Professor of Microbiology Department of Microbiology, National Center for Radiation Research and Technology, Nasr City, Cairo for her great active continuous help in the theoretical and practical parts of this work and for her direct supervision. I wish to thanks also Prof. Dr. Nagy Halim Aziz Professor of Microbiology Department of Microbiology, National Center for Radiation Research and Technology, Nasr City, Cairo for his encouragement. I wish also to express my deepest thanks to every body else who contributed in any way in this work. vi Aim of the Work Dedication To The Spirit of my Father vii Aim of the Work LIST OF CONTENTS Title Page No. Introduction ........................................................................................ i Aim of hte Work ................................................................................. iii 1. Literature review ............................................................................ 1 1.1. Spreading of polycyclic aromatic hydrocarbons (PAHs) in nature ........................................................................ 1 1.2. Health impact of PAHs .............................................................. 7 1.3. Petroleum oil contamination ...................................................... 23 1.4. Polycyclic aromatic hydrocarbons as constituent of petroleum contaminants .............................................................. 24 1.5. Microorgnaisms degrading PAHs. ............................................. 34 1.6. Mechanism of PAHs degradation strains .................................... 42 1.7. Pathway for PAHs degradation .................................................. 53 2. Materials and Methods ............................................................ 67 2.1. Materials ................................................................................... 67 2.2. Methods ..................................................................................... 73 3. Results and discussion ............................................................. 80 3.1. Growth of different indigenous bacterial communities on different PAHs ....................................................................... 80 3.2. Determination of the total bacterial count and the hydrocarbon degrading bacteria (HDB) found in each community ................................................................................. 111 3.3. Isolation and determination of the most potent strains having the ability to degrade different PAHs............................... 113 3.4. Growth and degradation of naphthalene by the most potent isolated strains ................................................................ 118 3.5. Growth and degradation of phenanthrene by the most potent isolated strains ................................................................ 135 3.6. Growth and degradation of anthracene by the most potent isolated strains ................................................................. 155 viii Aim of the Work LIST OF CONTENTS (Cont…) Title Page No. 3.7. Growth and degradation of pyrene by the most potent isolated strains ............................................................................ 189 3.8. Growth and degradation of benzo-a-anthracene by the most potent isolated strains ......................................................... 207 3.9. Identification of the most potent PAHs degrading bacterial strains .......................................................................... 224 3.10. Effect of gamma radiation on the viability of Bacillus amyloliquefaciens ...................................................................... 230 3.11. Selection of the hyper PAHs degrading bacterial mutant ....................................................................................... 232 3.12. Pathway of B. amyloliquefaciens for degradation of PAHs compounds. ...................................................................... 236 Summary ............................................................................................ 256 References .......................................................................................... 260 Arabic Summary ................................................................................ ––– ix Aim of the Work LIST OF FIGURES Fig. No. Title Page No. Figure (1): Proposed pathway for the degradation of naphthalene by Pseudomonas putida.......................... 55 Figure (2): Proposed pathway for the degradation of naphthalene by Streptomyces griseus ......................... 55 Figure (3): Proposed pathway for the degradation of phenanthrene by Sphingomonas sp............................. 58 Figure (4): Postulated metabolic pathway of PAH- degradation in aerobic bacteria .................................. 58 Figure (5): Proposed phenanthrene degradation pathways by the managrove enriched bacterial consortium .................................................. 59 Figure (6): Proposed pathway for the degradation of phenanthrene by pleurotus ostreatus .......................... 60 Figure (7): Proposed pathway for the degradation of anthracene by Aspergillus fumigatus .......................... 62 Figure (8): Proposed pyrene degradation pathways by the mangrove enriched bacterial consortium ................................................................. 64 Figure (9): Proposed pathway for the degradation of pyrene by Mycobacterium flavescens .......................... 65 Figure (10): Proposed pathway for the degradation of pyrene by Mycobacterium sp. strain PYR- 1 ................................................................................ 65 Figure (11): Proposed pathway for the degradation of pyrene by Aspergillus niger SK 93/7 ......................... 66 Figure (12): Proposed pathway of benzo[a]anthracene degradation by the ligninolytic fungus Irpex lacteus .............................................................. 67 Figure (13): Proposed pathways for the degradation of [B-a-Anth.] by Mycobacterium sp. strain RJGII-135 based on isolated metabolites ................... 67 Figure (14): Sampling site map....................................................... 69 x Aim of the Work LIST OF FIGURES (Cont…) Fig. No. Title Page No. Figure (15): Growth of different indigenous bacterial communities on 500mg/l naphthalene. ....................... 88 Figure (16): Concentration of extracellular protein of different bacterial communities communities on 500mg/l naphthalene. ....................... 88 Figure (17): Growth of different indigenous bacterial communities on 250mg/l phenanthrene. ..................... 90 Figure (18): Concentration of extracellular protein of different indigenous bacterial communities on 250mg/l phenanthrene. .......................................... 90 Figure (19): Growth of different indigenous bacterial communities on 50mg/l anthracene. ........................... 93 Figure (20): Concentration of extracellular protein of different indigenous bacterial communities on 50mg/l anthracene. ................................................ 93 Figure (21): Growth
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