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Aim of the Work

BIODEGRADATION OF POLYCYCLIC AROMATIC IN 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 ()

Botany Department Faculty of Science Cairo University

(2013)

i Aim of the Work

ABSTRACT

Student Name: Abir Moawad Partila Girgis

Title of the thesis: of Polycyclic Aromatic 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 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 Bacillus amyloliqueficiens have been exposed to different doses of gamma radiation to improve its qualities.

Keywords: Polycyclic- Aromatic- Hydrocarbon – Biodegradation-.

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 ...... 1 1.2. Health impact of PAHs ...... 7 1.3. Petroleum oil ...... 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 (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 by the most potent isolated strains ...... 118 3.5. Growth and degradation of by the most potent isolated strains ...... 135 3.6. Growth and degradation of 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 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 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 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 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): of extracellular 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 of different indigenous bacterial communities on 100mg/l ...... 95 Figure (22): Concentration of extracellular protein of different indigenous bacterial communities on 100mg/l acenaphthene...... 95 Figure (23): Growth of different indigenous bacterial communities on 10mg/l fluoranthene...... 98 Figure (24): Concentration of extracellular protein of different indigenous bacterial communities on 10mg/l fluoranthene...... 98 Figure (25): Growth of different indigenous bacterial communities on 100ug/l pyrene...... 100 Figure (26): Concentration of extracellular protein of different indigenous bacterial communities on 100 ug/l pyrene...... 100

xi Aim of the Work

LIST OF FIGURES (Cont…)

Fig. No. Title Page No.

Figure (27): Growth of different indigenous bacterial communities on 100ug/l benzo-a- anthracene...... 103 Figure (28): Concentration of extracellular protein of different indigenous bacterial communities on 100ug/l benzo-a-anthracene...... 103 Figure (29): Count of different indigenous bacterial communities on different polycyclic aromatic hydrocarbons (PAHs) after 28 days incubation...... 107 Figure (30): Count of different indigenous bacterial communities on different media...... 115 Figure (31): Growth of strain MAM-26 on different concentrations of naphthalene...... 123 Figure (32): Extracellular protein of strain MAM-26 on different concentrations of naphthalene...... 123 Figure (33): Growth of strain MAM- 43 on different concentrations of naphthalene...... 126 Figure (34): Extracellular protein of strain MAM- 43 on different concentrations of naphthalene...... 126 Figure (35): Growth of strain MAM- 62 on different concentrations of naphthalene...... 129 Figure (36): Extracellular protein of strain MAM-62 on different concentrations of naphthalene...... 129 Figure (37): Growth of strain MAM- 68 on different concentrations of naphthalene...... 131 Figure (38): Extracellular protein of strain MAM-68 on different concentrations of naphthalene...... 131 Figure (39): Growth of strain MAM-78 on different concentrations of naphthalene...... 134

xii Aim of the Work

LIST OF FIGURES (Cont…)

Fig. No. Title Page No.

Figure (40): Extracellular protein of strain MAM-78 on different concentrations of naphthalene...... 134 Figure (41): Degradation percentage of naphthalene after 21 days by HPLC...... 139 Figure (42): Growth of strain MAM- 26 on different concentrations of phenanthrene...... 141 Figure (43): Extracellular protein of strain MAM-26 on different concentrations of phenanthrene...... 141 Figure (44): Growth of strain MAM-43 on different concentrations of phenanthrene...... 144 Figure (45): Extracellular protein of strain MAM-43 on different concentrations of phenanthrene...... 144 Figure (46): Growth of strain MAM-62 on different concentrations of phenanthrene...... 147 Figure (47): Extracellular protein of strain MAM- 62 on different concentrations of phenanthrene...... 147 Figure (48): Growth of strain MAM- 68 on different concentrations of phenanthrene...... 149 Figure (49): Extracellular protein of strain MAM-68 on different concentrations of phenanthrene...... 149 Figure (50): Growth of strain MAM- 78 on different concentrations of phenanthrene...... 152 Figure (51): Extracellular protein of strain MAM-78 on different concentrations of phenanthrene...... 152 Figure (52): Degradation percentage of phenanthrene after 21 days by HPLC...... 156 Figure (53): Growth of strain MAM-26 on different concentrations of anthracene...... 160

xiii Aim of the Work

LIST OF FIGURES (Cont…)

Fig. No. Title Page No.

Figure (54): Extracellular protein of strain MAM-26 on different concentrations of anthracene...... 160 Figure (55): Growth of strain MAM-29 on different concentrations of anthracene...... 163 Figure (56): Extracellular protein of strain MAM-29 on different concentrations of anthracene...... 163 Figure (57): Growth and extracellular protein of strain MAM-43 on different concentrations of anthracene...... 165 Figure (58): Extracellular protein of strain MAM-43 on different concentrations of anthracene...... 165 Figure (59): Growth of strain MAM-62 on different concentrations of anthracene...... 168 Figure (60): Extracellular protein of strain MAM-62 on different concentrations of anthracene...... 168 Figure (61): Growth of strain MAM-68 on different concentrations of anthracene...... 170 Figure (62): Extracellular protein of strain MAM-68 on different concentrations of anthracene...... 170 Figure (63): Growth of strain MAM- 78 on different concentrations of anthracene...... 173 Figure (64): Extracellular protein of strain MAM-78 on different concentrations of anthracene...... 173 Figure (65): Growth of strain E. cloacae MAM -4 on different concentrations of anthracene...... 175 Figure (66): Extracellular protein of strain E.cloacae MAM-4 on different concentrations of anthracene...... 175 Figure (67): Growth of strain MAM-26 on higher concentrations of anthracene...... 178

xiv Aim of the Work

LIST OF FIGURES (Cont…)

Fig. No. Title Page No.

Figure (68): Extracellular protein of strain MAM-26 on higher concentrations of anthracene...... 178 Figure (69): Growth of strain MAM-29 on higher concentrations of anthracene...... 180 Figure (70): Extracellular protein of strain MAM-29 on higher concentrations of anthracene...... 180 Figure (71): Growth of strain MAM- 62 on higher concentrations of anthracene...... 183 Figure (72): Extracellular protein of strain MAM-62 on higher concentrations of anthracene...... 183 Figure (73): Growth of strain MAM-68 on higher concentrations of anthracene...... 185 Figure (74): Extracellular protein of strain MAM-68 on higher concentrations of anthracene...... 185 Figure (75): Growth of strain E.cloacae MAM-4 on higher concentrations of anthracene...... 188 Figure (76): Extracellular protein of strain E.cloacae MAM-4 on higher concentrations of anthracene...... 188 Figure (77): Degradation percentage of anthracene after 21 days by HPLC...... 191 Figure (78): Growth of strain MAM-26 on different concentrations of pyrene...... 194 Figure (79): Extracellular protein of strain MAM-26 on different concentrations of pyrene ...... 194 Figure (80): Growth of strain MAM-29 on different concentrations of pyrene...... 197 Figure (81): Extracellular protein of strain MAM-29 on different concentrations of pyrene...... 197

xv Aim of the Work

LIST OF FIGURES (Cont…)

Fig. No. Title Page No.

Figure (82): Growth of strain MAM- 62 on different concentrations of pyrene...... 199 Figure (83): Extracellular protein of strain MAM-62 on different concentrations of pyrene...... 199 Figure (84): Growth of strain MAM-68 on different concentrations of pyrene...... 202 Figure (85): Extracellular protein of strain MAM-68 on different concentrations of pyrene...... 202 Figure (86): Growth of strain E.cloacae MAM-4 on different concentrations of pyrene...... 204 Figure (87): Extracellular protein of strain E.cloacae MAM-4 on different concentrations of pyrene...... 204 Figure (88): Degrdation percentage of Pyrene after 21 days by HPLC...... 209 Figure (89): Growth of strain MAM-26 on different concentrations of benzo-a-anthracene...... 213 Figure (90): Extracellular protein of strain MAM-26 on different concentrations of benzo-a- anthracene...... 213 Figure (91): Growth of strain MAM-29 on different concentrations of benzo-a-Anthracene...... 215 Figure (92): Extracellular protein of strain MAM-29 on different concentrations of benzo-a- anthracene...... 215 Figure (93): Growth of strain MAM-62 on different concentrations of benzo-a-anthracene...... 217 Figure (94): Extracellular protein of strain MAM- 62 on different concentrations of benzo-a- anthracene...... 217

xvi Aim of the Work

LIST OF FIGURES (Cont…)

Fig. No. Title Page No.

Figure (95): Growth of strain MAM- 68 on different concentrations of benzo-a-anthracene...... 220 Figure (96): Extracellular protein of strain MAM-68 on different concentrations of benzo-a- anthracene...... 220 Figure (97): Growth of strain E.cloacae MAM-4 on different concentrations of benzo-a- anthracene...... 222 Figure (98): Extracellular protein of strain E.cloacae MAM-4 on different concentrations of benzo-a-anthracene...... 222 Figure (99): Degradation percentage of benzo-a- anthracene after 21 days by HPLC...... 226 Figure (100): Agarose gel of DNA of isolated strains MAM-29 and MAM-62 polycyclic aromatic hydrocarbon degrading bacteria ...... 228 Figure (101): DNA sequencing of isolate MAM-29...... 229 Figure (102): Phylogenetic tree constructed to isolated strain MAM-29...... 229 Figure (103): DNA sequencing of isolate MAM-62...... 230 Figure (104): Phylogenetic tree constructed to isolated strain MAM-62...... 230 Figure (105): Effect of gamma-radiation doses on the viable count of isolated strain MAM-62 ...... 234 Figure (106): Proposed pathway for the degradation of naphthalene by B. amyloliquefaciens MAM-62...... 234 Figure (107): Proposed pathway for the degradation of naphthalene by the mutant of B. amyloliquefaciens MAM-62(4)...... 240

xvii Aim of the Work

LIST OF FIGURES (Cont…)

Fig. No. Title Page No.

Figure (108): Proposed pathway for the degradation of phenanthrene by B. amyloliquefaciens MAM-62...... 243 Figure (109): Proposed pathway for the degradation of phenanthrene by the mutant of B. amyloliquefaciens MAM-62(4)...... 244 Figure (110): Proposed pathway for the degradation of anthracene by B. amyloliquefaciens and it's mutant MAM-62(4)...... 247 Figure (111): Proposed pathway for pyrene degradation by B. amyloliquefaciens MAM-62 and it's mutant MAM-62(4)...... 250 Figure (112): Proposed pathway of benzo-a-anthracene degradation by B. amyloliquefaciens MAM-62 ...... 253 Figure (113): Proposed pathway for benzo-a-anthracene degradation by the mutant of B. amyloliquefaciens MAM-62(4)...... 254

xviii Aim of the Work

LIST OF TABLES

Tab. No. Title Page No. Table (1): Sampling Sites represented different sources of indigenous microbial communities ...... 70 Table (2): Growth and extracellular protein of different indigenous bacterial communities on 500 mg/L naphthalene...... 87 Table (3): Growth and extracellular protein of different indigenous bacterial communities on 250 mg/l phenanthrene...... 89 Table (4): Growth and extracellular protein of different indigenous bacterial communities on 50 mg/l anthracene...... 92 Table (5): Growth and extracellular protein of different indigenous bacterial communities on 100 mg/l acenaphthene...... 94 Table (6): Growth and extracellular protein of different indigenous bacterial communities on 10 mg/l fluoranthene...... 97 Table (7): Growth and extracellular protein of different indigenous bacterial communities on 100 ug/l pyrene...... 99 Table (8): Growth and extracellular protein of different indigenous bacterial communities on 100 ug/l benzo-a-anthracene...... 102 Table (9): Count of different indigenous bacterial communities on different polycyclic aromatic hydrocarbons (PAHs) after 28 days incubation. 105 Table (10): Increase of indigenous bacterial communities count after 28 days of incubation on different PAHs...... 106 Table (11): Count of different indigenous bacterial communities on different media...... 115

xix Aim of the Work

LIST OF TABLES (Cont…) Tab. No. Title Page No.

Table (12): The ability of indigenous isolated strains to grown on different PAHs at different concentrations...... 117 Table (13): Characters of isolated strains on BSM agar supplemented with PAHs compounds...... 119 Table (14): Growth and extracellular protein of strain MAM- 26 on different concentrations of naphthalene...... 122 Table (15): Growth and extracellular protein of strain MAM- 43 on different concentrations of naphthalene...... 125 Table (16): Growth and extracellular protein of strain MAM- 62on different concentrations of naphthalene...... 128 Table (17): Growth and extracellular protein of strain MAM-68 on different concentrations of naphthalene...... 130 Table (18): Growth and extracellular protein of strain MAM-78 on different concentrations of naphthalene...... 133 Table (19): Count of the selected isolated strains on different concentrations of naphthalene after 21 days incubation period...... 135 Table (20): Degradation percentage of naphthalene after 21 days by HPLC...... 139 Table (21): Growth and extracellular protein of strain MAM-26 on different concentrations of phenanthrene...... 140 Table (22): Growth and extracellular protein of strain MAM-43 on different concentrations of phenantherene ...... 143

xx Aim of the Work

LIST OF TABLES (Cont…)

Tab. No. Title Page No.

Table (23): Growth and extracellular protein of strain MAM-62 on different concentrations of phenanthrene...... 146 Table (24): Growth and extracellular protein of strain MAM-68 on different concentrations of phenanthrene...... 148 Table (25): Growth and extracellular protein of strain MAM-78 on different concentrations of phenanthrene...... 151 Table (26): Count of the selected isolated strain on different concentrations of phenanthrene after 21 days incubation period...... 153 Table (27): Degradation percentage of phenanthrene after 21 days by HPLC...... 156 Table (28): Growth and extracellular protein of strain MAM- 26- on different concentrations of anthracene...... 159 Table (29): Growth and extracellular protein of strain MAM-29 on different concentrations of anthracene...... 162 Table (30): Growth and extracellular protein of strain MAM-43 on different concentrations of anthracene...... 164 Table (31): Growth and extracellular protein of strain MAM-62 on different concentrations of anthracene...... 167 Table (32): Growth and extracellular protein of strain MAM-68 on different concentrations of anthracene...... 169

xxi Aim of the Work

LIST OF TABLES (Cont…)

Tab. No. Title Page No.

Table (33): Growth and extracellular protein of strain MAM-78 on different concentrations of anthracene...... 172 Table (34): Growth and extracellular protein of strain E. cloacae MAM-4 on different concentrations of anthracene...... 174 Table (35): Growth and extracellular protein of strain MAM-26 on higher concentrations of anthracene...... 177 Table (36): Growth and extracellular protein of strain MAM-29 on higher concentrations of anthracene...... 179 Table (37): Growth and extracellular protein of strain MAM-62 on higher concentrations of anthracene...... 182 Table (38): Growth and extracellular protein of strain MAM-68 on higher concentrations of anthracene...... 184 Table (39): Growth and extracellular protein of strain E.cloacae MAM- 4 on higher concentrations of anthracene. 187 Table (40): Count of the selected isolated strains on different concentrations of anthracene after 21 days incubation period...... 190 Table (41): Degradation percentage of anthracene after 21 days by HPLC...... 191 Table (42): Growth and extracellular protein of strain MAM-26 on different concentrations of pyrene...... 193

xxii Aim of the Work

LIST OF TABLES (Cont…)

Tab. No. Title Page No.

Table (43): Growth and extracellular protein of strain MAM-29 on different concentrations of pyrene...... 196 Table (44): Growth and extracellular protein of strain MAM-62on different concentrations of pyrene...... 198 Table (45): Growth and extracellular protein of strain MAM-68 on different concentrations of pyrene...... 201 Table (46): Growth and extracellular protein of strain E.cloacae MAM-4 on different concentrations of pyrene...... 203 Table (47): Count of the selected isolated strains on different concentrations of pyrene after 21 days incubation...... 205 Table (48): Degrdation percentage of Pyrene after 21 days by HPLC...... 209 Table (49): Growth and extracellular protein of strain MAM-26 on different concentrations of benzo- a-anthracene (B-a-Anth.)...... 212 Table (50): Growth and extracellular protein of strain MAM-29 on different concentrations of benzo-a-Anthracene...... 214 Table (51): Growth and extracellular protein of strain MAM- 62 on different concentrations of benzo-a-anthracene (B-a-Anth)...... 216 Table (52): Growth and extracellular protein of strain MAM- 68 on different concentrations of benzo- a-anthracene (B-a-Anth.)...... 219 Table (53): Growth and extracellular protein of strain E.cloacae MAM- 4 on different concentrations of benzo-a-anthracene (B-a- Anth.)...... 221 xxiii Aim of the Work

LIST OF TABLES (Cont…)

Tab. No. Title Page No.

Table (54): Count of the selected isolated strains on different concentrations of benzo-a- anthracene after 21 days incubation period...... 224 Table (55): Degradation percentage of benzo-a- anthracene after 21 days by HPLC...... 226 Table (56): Effect of gamma irradiation on the viability of B. amyloliquefaciens...... 234 Table (57): Growth of the parent strain of B. amyloliquefaciens and its different selected mutants isolated exposed to different doses of gamma radiation on different PAHs...... 236 Table (58): The increase in growth of the selected mutants (I)on different PAHs after different incubation periods compared to the parent strain(Io) ...... 238 Table (59): Intermediates determined by GC-MS analysis of Naphthalene degradation by B. amyloliquefaciens and its mutant MAM- 62(4) after 24 hours incubation...... 240 Table (60): Intermediates determined by GC-MS analysis of Phenanthrene degradation by B. amyloliquefaciens MAM-62 and its mutant MAM-62(4) after 24 hours incubation...... 245 Table (61): Intermediates determined by GC-MS analysis of anthracene degradation by B. amyloliquefaciens MAM-62 and its mutant MAM-62(4) after 24 hours incubation...... 249 Table (62): Intermediates determined by GC-MS analysis of pyrene degradation by B. amyloliquefaciens MAM-62 and its mutant MAM-62(4) after 24 hours incubation...... 252 Table (63): Intermediates determined by GC-MS analysis of Benzo-a-anthracene degradation by B. amyloliquefaciens MAM-62 and its mutant MAM-62(4) after 24 hours incubation...... 255

xxiv Aim of the Work

LIST OF ABBREVIATION

Abbrev. Full term

Ace Acenaphthene. Anth Anthracene. B-aAnth Benzo-a-anthracene. Blastn Somewhat similar sequences. BSM Basal medium. CFU Cell Forming Unit. Flu Fluoranthene. GC-MS / Spectrometry. HDB Hydrocarbon Degrading Bacteria. HPLC High Performance Chromatography. KGy KiloGray LBL Uria-Bertani broth medium. Mg/L Milligram per liter. Naph. Naphthalene. NCBI National Center for Information. NCRRT National Center for Radiation Research and PAH Polycyclic aromatic hydrocarbon. PAHDB Polycyclic aromatic hydrocarbon Degrading Phen. Phenanthrene. Pyr. Pyrene. TE buffer -HCl buffer used to store DNA and RNA Technology. Ug/L Microgram per liter. Ug/ml Microgram per milliliter

xxv Aim of the Work

INTRODUCTION

xxvi Aim of the Work

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants in urban (Chen et al., 2013).

PAHs enter the environment via incomplete of fossil fuels and accidental leakage of petroleum products, and as components of products such as (Muckian et al., 2009).

Due to PAHs carcinogenic activity, they have been included in the European Union (EU) and the Environmental Protection Agency (EPA) priority pollutant lists. exposure to PAHs occurs in three ways, inhalation, dermal contact and consumption of contaminated , which account for 88–98% of such contamination; in other words, diet is the major source of human exposure to these contaminants (Rey-Salgueiro et al., 2008).

Both the World Health Organization and the UK Expert Panel on Air Quality Standards (EPAQS) have considered benzo(a)pyrene (BaP) as a marker of the carcinogenic potency of the polycyclic aromatic hydrocarbons (PAH) mixture (Delgado-Saborit et al., 2011).

Polycyclic aromatic and heavier aliphatic hydrocarbons, which have a stable recalcitrant molecular structure, exhibit high hydrophobicity and low aqueous , are not readily removed from through leaching and volatilization (Brassington et al., 2007).

The hydrophobicity of PAHs limits desorption to the aqueous phase (Donlon et al., 2002). Six main ways of dissipation, i.e. disappear- ance, are recognized in the environment: volatilization, photooxidation,

xxvii Aim of the Work chemical oxidation, sorption, leaching and biodegradation. Microbial degradation is considered to be the main process involved in the dissipation of PAH (Yuan et al., 2002).

Thus, more and more research interests are turning to the biodegradation of PAHs. Some can utilize PAHs as a source of and energy so that PAHs can be degraded to and , or transformed to other nontoxic or low-toxic substances (Perelo, 2010).

Compared with other physical and chemical methods such as combustion, photolysis, landfill and ultrasonic , biodegradation is expected to be an economic and environmentally friendly alternative for removal of PAHs (Toledo et al., 2006).

Márquez-Rocha et al. (2005) revealed that many isolated bacterial and fungal have been reported to be capable of biodegrading effectively petroleum hydrocarbons and even polynuclear aromatic hydrocarbons.

xxviii Aim of the Work

AIM OF THE WORK

The over all objective of this study is to degrade the serious hazardous , low and high molecular weight (LMW and HMW) polycyclic aromatic hydrocarbons (PAHs) which are carcinogenic and mutagenic by ecofriendly manner via indigenous bacterial communities, and getting the most potent strains able to degrade different PAHs isolated from the indigenous bacterial communities of petroleum oil contaminated samples.

This main objective can be divided to subobjectives:

1- Getting indigenous bacterial communities able to utilize PAHs as sole carbon and energy source.

2- Isolation of hydrocarbon degrading bacteria (HDB).

3- Isolation of polycyclic aromatic hydrocarbon degrading bacterial strains (most potent strains).

4- Studying the abilities of the isolated bacterial strains to grow and degrade different PAHs with different concentrations.

5- Identification of the most potent strains by 16SrRNA.

6- Improving the ability of the most promising selected strain to degrade PAHs by gamma radiation.

7- Compairing the degradation proposed pathways of the parent strain (wild type) with the mutant strain.

xxix Literature Review

LITERATURE REVIEW

1 Literature Review

1. LITERATURE REVIEW

1.1 Spreading of polycyclic aromatic hydrocarbons (PAHs) in nature:

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants found in all environmental compartments. Sources vary widely from natural to anthropogenic (Harvey, 1997). Due to their ubmiquitous nature, PAHs are found in a wide range of environments including , sediments, ground , and the (Trapido, 1999). It is estimated that most of the total environmental PAH load (90%) is found in terrestrial , and more specifically, the top 20 cm of the soil horizon (Maliszewska- Kordybach, 1999).

Basu et al., (1987) revealed that in aquatic environments, PAHs tend to adsorb to particulate and most PAH contamination in these environments is concentrated in sediments, or associated with the presence of suspended solids in surface waters.

More than 2800 chemicals have been identified in the ambient air including polycyclic aromatic hydrocarbons (PAHs), nitrated and halogenated organic compounds, derivatives, or (Lewtas and Gallagher, 1990).

Liu et al., (2003) showed that PAHs from combustion mainly occur in floating dusts and flues.

The incomplete combustion results in a clear increase in PAH emission compared with other combustion methods; this is attributed to the formation of species with two and three rings, such as naphthalene, acenaphthylene, and acenaphthene. Benzo[a] pyrene, dibenz[a,h]anthracene, and benz[a]anthracene make a large contribution to the equivalent value (TEQ) (Liu et al., 2012). 2 Literature Review

1.1.1. Contamination of soils:

Polycyclic aromatic hydrocarbons (PAHs) extensively occur as pollutants in soil and water, and are important environmental contaminants because of their recalcitrance (Deziel et al., 1996).

PAHs present in soil may exhibit a toxic activity towards different , microorganisms and invertebrates (Schloter et al., 2003).

Different organic compounds present in , fungicides, detergents, and mothballs contain certain PAHs like naphthalene and phenanthrene (Samanta et al., 2002; Morzik et al., 2003 and Johnsen et al., 2005). Polycyclic aromatic hydrocarbons (PAHs) often found in high residual soil concentrations at industrial sites (Parrish et al., 2005).

Yang et al., (2005) found that soils as reservoirs receive a large amount of PAHs and some of them are carcinogenic and/or mutagenic and may pose threats to human health.

Sewage sludge addition to soils resulted in an increase in the content of polycyclic aromatic hydrocarbons in these soils (Oleszczuk, 2006). Anthropogenic hydrocarbon contamination of soil is a global issue throughout the industrialised world (Brassington et al., 2007).

Pizzul et al., (2007) indicated that PAH are often found in contaminated soils and there is the need of developing techniques that can be applied in the remediation of these sites, where PAH, specially those with high molecular weight, pose health and environmental risks.

Vaughan (1984) indicated that atmospheric deposition on leaves often greatly exceeds uptake from soil by roots as a route of PAH accumulation. Pine needles were used as passive samplers in assessing ambient atmospheric concentrations of persistent organic contaminants,

3 Literature Review such as PAHs and dichloro benzenepdioxins on regional and global scales (Tremolada et al., 1996).

Compared with PAHs dissolved in water, it was found that PAHs sorbed on pine needles had low photolysis rates, thus suggesting that the of the pine needles can stabilize PAH photolysis (Wang et al., 2005).

Gaseous diffusion from the air to the waxy layer of leaves has been shown to be a major uptake process for these lipophilic organic contaminants (Wild et al., 2005, 2006).

The effect and fate of polycyclic aromatic hydrocarbons (PAHs) in nature are of great environmental and human health concerns due to their widespread occurrence, persistence in terrestrial ecosystems and carcinogenic properties and has led to numerous studies on contaminated soils and also to different approaches for remediation of soil PAH pollutants (Mastrangela et al., 1996; Maynard et al., 1997 Goldman et al., 2001; Johnsen et al., 2005 and Liste and Prutz, 2006).

4 Literature Review

1.1.2. Contamination of water:

Large-scale oil spills have significant impacts on both and human society (Hayakawa et al., 1997). Severe subsurface pollution of soils and water can occur via the leakage of underground storage tanks and pipelines, spills at production wells and distribution terminals, and seepage from gasworks sites during coke production, contributing as a major organic contamination to the natural environment (Juck et al., 2000 and Bundy et al., 2002).

Yuan et al., (2001) found that among organic pollutants, polycyclic aromatic hydrocarbons (PAHs) are common in fresh water ecosystems and particularly in river sediments where they accumulate. Crustacean heart rate is a useful biomarker. Cardiac activity increased in Carcinus maenas following exposure to the water soluble fraction of crude oil (Depledge, 1984).

Pilot-scale constructed were used to treat water contaminated by polycyclic aromatic hydrocarbons (PAHs), particularly fluoranthene (Giraud et al., 2001).

Recent increases in the aquatic accumulation of PAHs over the last decade have been detected and are associated with increased use of motor vehicles (Lima et al., 2002).

Maskaoui et al., (2002) showed that the levels of benzo(a) pyrene (BaP), pyrene(Py) and phenanthrene (Phe) in the surface water from the Jiulong River Estuary and Western Xiamen Sea were 0.56-3.32, 0.22- 2.19and 0.16-1.37 mg L-1, respectively. River sediments contain various and organic constituents of natural and anthropogenic origin that are or may become potential pollutants (Jaffé et al., 2003).

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It is reported that concentrations of benzo[a]pyrene (BaP) vary from 1.0 to 23.4 ng L-1 in the surface sea water of Maluan Bay in Xiamen, China, whereas the concentration of total PAHs are 5.118 mg g-1 in the sediments of Xiamen western harbor, China (Tian et al., 2004). PAHs are lipophilic organic compounds and widespread in the marine environment (HELCOM, 2009).

Means et al., (1980) reported that in marine environments, most PAHs do not dissolve well in water and tend to accumulate in sediments. As they enter the marine environment, PAHs bind tightly to suspended particles and consequently accumulate in bottom sediments due to their low water solubility and hydrophobic properties (Varanasi, 1989), these PAHs in the marine sediments are recalcitrant and persistent and thus have a strong tendency to become concentrated in marine webs.

Wild and Jones (1989) reported that wastewater analyses reveal high PAH concentrations from such sources as industrial waste, domestic , atmospheric rainfall, airborne pollutants, and road surface run-off. More soluble petroleum hydrocarbon (PHC) components such as monoaromatic hydrocarbons are continuously released from the source area into the ground water (Bedient et al., 1994).

Hughes et al., (1997) indicated that they are hydrophobic and readily adsorbed onto particulate matter, thus, coastal and marine sediments become the ultimate sinks for PAHs.

The largest fraction enters marine waters as land-based runoff or atmospheric deposition or oil spills (National Research Council, 2003).

Camus et al., (2002) found that increasing industrial activity in the European Arctic has raised concerns of the potential anthropogenic impact of chemicals on this polar marine .

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1.1.3. Contamination of air:

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the polluted atmospheric environment in the ngm-3 concentration range. The gaseous state is predominant for the lighter molecular weight PAHs, while the substances with more than 4rings are preferentially associated with the aerosol particles (ECPACWG, 2001).

Rogge et al., (1993) revealed that human spend more than 80% time in indoors. The quality of indoor air has an important impact on human health. However, there are many sources of PAHs in indoor air, such as heating/cooking and or electric stoves.

Sheldon et al., (1993) found that the concentrations of PAHs from cigarette were 1.5–4 times higher than that of other indoor combustion sources. One significant source of PAHs indoors is environmental tobacco (ETS) (Rogg et al., 1994).

Perera et al., (2005) found that Benzo[a]pyrene (BaP) is a representative member of polycyclic aromatic hydrocarbons (PAHs), which are combustion-related pollutants widely present in the environment. Further study indicated that more than 80% of BaP in indoor air of resident homes in Hangzhou was from (Lu and Zhu, 2007).

These compounds are known for their presence in the atmosphere, water, sediments, tobacco smoke and food ( Rey-Salgueiro et al., 2008). PAHs are ubiquitous in outdoor and indoor air (Harrison et al., 2009).

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1.2. Health impact of PAHs

Xenobiotic chemicals are continuously released into the biosphere, posting a significant risk to human health due to their toxicity and persistence in the environment. Polycyclic aromatic hydrocarbons (PAHs) from natural and/or anthropogenic sources are characterized by their teratogenic, mutagenic and carcinogenic properties (Blumer, 1976).

The Environmental Protection Agency (USEPA) lists 16 kinds of PAHs as priority pollutants (Kieth and Telliard, 1979). PAHs are highly toxic and several of these, including phenanthrene, and fluoranthene, are listed by the US Environmental Protection Agency (USEPA) as priority pollutants (USEPA, 1985 and White, 1986).

Hydrophobic organic contaminants (HOCs) are a class of ubiquitous compounds, which have posed high risk to the human health and ecological systems (Perera, 1997).

The contamination of PAHs and their derivatives in the environment, such as in soils, sediments, aerosols, water and organisms, is harmful to the health of both and ecosystems because they may cause mutagenic and carcinogenic effects (Lee and Gu, 2003).

Polycyclic aromatic hydrocarbons (PAHs) are an important class of ubiquitous environmental contaminants because of their high potential toxicity, mutagenicity and/or carcinogenicity (Mastrangela et al., 1996; Marston et al., 2001 and Xue and Warshawsky, 2005).

A target value of 1.0 ng/m3 with regard to Benzo(a)pyrene (BaP) for the total content in the particulate matter fraction averaged over a calendar year (Callén et al., 2011).

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1.2.1. On Human

The US Environmental Protection Agency has identified 16 unsubstituted PAHs as priority pollutants, eight of which are possible human (Heitkamp and Cerniglia, 1988; Menzi et al., 1992 and USEPA, 1998).

Madsen (1991) found that although many of these PAH compounds may undergo photolysis, chemical oxidation or volatilization, some may persist and, as result, accumulate in the environment, causing toxic, mutagenic, or carcinogenic effects.

Cerniglia (1992) showed that high-molecular-weight PAHs are important constituents of petroleum; and many are carcinogenic or mutagenic.

Lichtfouse et al. (1997) observed that the occurrence of polycyclic aromatic hydrocarbons (PAHs) in soils, sediments, aerosols, waters, animals and plants is of increasing environmental concern because some PAHs may exhibit mutagenic and carcinogenic effects. Since PAHs exhibit toxic, mutagenic and carcinogenic properties, there is serious concern about their environmental presence, especially their potential for bioaccumulation in many food chains (Fujikawa et al., 1993; Budavari, 1996 and Harvey, 1996). In the cell, PAH are oxidized by cytochrome P450sto form electrophilic derivatives (e.g., diolepoxides and cations) that react with DNA to form adducts (Cavalieri and Rogan, 1995).

PAHs have been recognised as a potential health risk due to their intrinsic chemical stability, high recalcitrance to different types of degradation and high toxicity to living organisms (Alexander, 1999). PAHs can enter into the interior of cell through dissolution, resulting in toxicity and mutation of living things (Zhu et al., 2000).

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As cells re-enter S-phase, the heteroduplex mutations are “fixed” in daughter DNA by one round of replication (Chakravarti et al., 2000).

Liu et al., (2001) found that polycyclic aromatic hydrocarbons (PAHs) are a well-known group of environmental pollutants. They are carcinogenic to human and mostly formed in combustion processes of organic materials. Soil acts as a repository for many hydrocarbons, which is a concern due to their adverse impact on human health and their environmental persistence (Semple et al., 2001).

Samanta et al., (2002) indicated that the existence of PAHs in nature is of great environmental concern owing to their toxic, mutagenic and carcinogenic properties. Soil massively contaminated with PAH represents considerable public health hazards.

Based upon the toxic equivalent principle for PAHs and WHO Unit risk factor for PAH, the exposure for PAH is associated with an increased risk of lung cancer among Bangkok residents (Ruchirawat et al., 2002).

16 PAHs suggested by US Environmental Protection Agency are currently determined in environmental samples. More recently, some other carcinogenic PAHs, including dibenzopyrenes, have been listed as the EU priority contaminants (EC, 2002).

Among atmospheric trace chemical substances, PAHs are considered to pose the highest human health risk (WHO, 2003).

Trans activation of the aryl hydrocarbon receptor(AhR) might therefore to deregulation of cell proliferation in epithelial cells, thus ultimately contributing also to tumour promotion. On the other hand, formation of DNA adducts might activate cellular defence mechanisms, including induction of apoptosis, cell cycle perturbation or DNA repair,

10 Literature Review which might further modify proliferative behaviour of cells( Chramostová et al., 2004).

However, numerous studies have proved that sludge contains a number of organic pollutants (Oleszczuk and Baran, 2004) which pose a potential-danger to human health.

PAH are environmental carcinogens (Yu and Campiglia, 2005), associated with skin, lung, pharynx, oral and other cancers (Hecht, 2002). In the cell, PAH–DNA adduct formation blocks replication and induces and excision repair (BER and NER) activities during a 24- h long period (Khan and Chakravarti, 2005).

Another non-genotoxic mode of action, which might affect carcinogenicity of dibenzo and benzochrysenes, could be perturbation of tissue homeostasis due to disruption of cell-to-cell communication (Trosko and Upham, 2005).

Many of PAHs have been found to exhibit cytotoxic, mutagenic and carcinogenic properties and therefore pose a serious risk to human health (Bamford and Singleton, 2005).

PAH react primarily with the bases in DNA, forming bulky stable and depurinating adducts (Cavalieri et al., 2005 and Todorovic et al., 2005).

Vondráček et al. (2006) showed that various types of polyaromatic compounds, including both unsubstituted PAHs and heterocyclic aromatics, are able to disrupt contact inhibition in liver epithelial cell model.

Due to their carcinogenic activity, PAHs have been included in the European Union (EU) and the Environmental Protection Agency (EPA) priority pollutant lists. Human exposure to PAHs occurs in three ways, inhalation, dermal contact and consumption of contaminated foods, which 11 Literature Review account for 88–98% of such contamination; in other words, diet is the major source of human exposure to these contaminants (Collins et al., 1991; Rey-Salgueiro et al., 2008 and Sharif et al., 2008).

The persistence of PAHs in the environment poses a potential threat to human health through bioaccumulation and biomagnifications via food chains (Wang et al., 2008).

Doyle et al., (2008) indicated that PAHs can enter human bodies through inhalation, ingestion, and skin contact. Exposures to PAHs have been linked to skin, lung, liver, intestine, and pancreas cancers.

Cleaning activities entail a certain risk of contact with the oil. Direct contact with these products can cause acute health problems, such as neurological disorders (headaches, nausea, dizziness, and somnolence) from volatile (VOC) exposure and breathing difficulty, digestive problems (nausea, vomiting, and abdominal pain), and skin and mucus problems due to PAHs (ATSDR, 1995 and Suarez et al., 2005).

A report on the 1997 Nakhodka spill in the Sea of showed that more than half of the males and 80% of the females who participated in cleanup operations suffered from acute disorders (Morita et al., 1999), mainly low back pain, headache, and eyes and throat inflammation. Similar results were observed in the Erika spill in 1999.

A survey found that 53% of the workers had reported at least one health problem, including headache, rash, eye redness, respiratory problems, nausea, and abdominal pain (Schvoerer et al., 2000). In Galicia, registered mainly eye redness, headache, sore throat, trauma, nausea, dizziness, and breathing difficulty (Conselleria de Sanidade, 2003).

Müncnerová and Agustin (1994) found that although some low- molecular-weight PAHs, such as the tricyclic anthracene, are not

12 Literature Review carcinogenic, their oxidation mechanisms are of considerable interest as the same arrangements of fused aromatic rings are found in the more complex carcinogenic PAHs, such as benzo [a]pyrene and benz[a]anthracene.

PAHs are reported to disturb the antioxidant defense system and responsible to induce oxidative stress. It is well known that, PAHs are not known to exhibit acute symptoms; metabolic activation of PAHs by cytochrome P 450 (CYP) 1A1-catalyzed reactions generates electrophilic metabolites and other reactive species (ROS), which tends to bind covalently with DNA and also cause interference with cell homeostasis (Li et al., 1996; Kim and Lee, 1997 and Shi et al., 2005 a,b).

Under physiological conditions, the normal production of ROS is matched by several cellular mechanisms. These mechanisms mainly consist of antioxidant molecules and scavenger , which is an important reactive oxygen removal system in the body of aerobic organisms. However, when reactive oxygen species (ROS) generation exceeds the capacity of the cellular antioxidants, it will cause oxidative stress and significant oxidative damage (Burdon, 1995; Duthie et al., 1996; Matés, 2000 and Greenwell et al., 2002).

The carcinogenic PAHs found to be associated with particles are metabolized to reactive molecules that can react with DNA to form bulky- DNA adducts. DNA adducts tend to be higher among subjects heavily exposed to urban and occupational pollutants (Peluso et al., 2001). PAH- DNA adducts have also been detected in the blood from newborns, whose mothers were living in polluted areas of Poland and China. The adduct level was similar in mothers and in the child (Perera et al., 2005). They suggesting that carcinogenic agents present in ambient can pass the placental barrier and initiate damage in the unborn child that is relevant for carcinogenesis. A positive association has been established between the level of PAH in ambient air and the bulky adduct level at medium to high

13 Literature Review level of PAH, but not at the low level situation generally observed at ambient pollution. Particles generated by combustion are composed of a carbon core to which other compounds such as metals and PAH adhere. The particles do induce oxidative stress mediated by a particle-induced inflammation causing macrophages to release ROS. (Srensen et al., 2003).

Many diseases that have common origin in oxidative stress being in childhood (Stewart et al., 2002 and Singh et al., 2008).

Lung cancer is expected to cause 10 million deaths per year worldwide by the year 2030 (Proctor, 2001).

PAH form stable and depurinating DNA adducts in mouse skin to induce preneoplastic mutations depurinating adducts play a major role in forming the tumorigenic mutations (Chakravarti et al., 2008).

Rietjens and Alink (2003) and Sofi et al., (2008) reported that in relation to the diet, this plays a very important role in the etiology and prevention of cancer, and other serious cardiovascular and neurodegenerative diseases.

Chiang and Liao, (2006) and Chiang et al., (2009) had provided evidence that 90% probabilities exposure to smoke emitted from heavy incense burning may promote lung cancer risk.

Exposure to environmental cigarette smoke poses significant risks for cancers and a of respiratory and cardiovascular diseases (OEHHA, 2005). Passive smoking has been recognized as a major cause for female lung cancer in Taiwan, where less than 5% of women are smokers. Studies showed that spousal smoking increased lung cancer risk 2.1 times to nonsmoking women in Taiwan, much higher than a 20% excess risk observed in the Western countries (IARC, 2004). A number of polycyclic aromatic hydrocarbons (PAHs) found in cigarette smoke of US

14 Literature Review and European brands, such as benz[a]-anthracene and benoz[a]pyrene,have been classified as carcinogens by the International Agency for Research on Cancer (IARC, 2010). Some of these compounds, especially those present in the particulate phase, exhibit a wide range of adverse effects on human health such as cardio-respiratory disorders (Dominici et al., 2006 and Hales and Howden-Chapman, 2007) or lung cancer mortality (Pope 3rd et al., 2002; Lewtas, 2007 and Lee et al., 2011).

Binding of polycyclic aromatic hydrocarbons (PAHs) with DNA is one of the key steps in their mutagenic process (Wang et al., 2009).

Knafla et al., (2011) showed that humans may be dermally exposed to the carcinogenic substance benzo[a] pyrene (B[a]P) via contact with soil at contaminated sites. the formation of epidermal tumors may be a more sensitive endpoint than systemic tumors following dermal exposure. B[a]P-related skin cancer (point of contact) risks should be considered at contaminated sites.

Pyrene is a toxic, recalcitrant, four fused ring PAH commonly found in soil (Saraswathy and Hallberg, 2005); its -based metabolites are mutagenic and more toxic than the parent compound (Singh, 2006). For these reasons, pyrene is listed among the 16 USEPA priority pollutants PAHs and considered as an indicator for monitoring PAH contaminated wastes (Saraswathy and Hallberg, 2005).

Anthracene is a three-ring PAH with relatively serious toxicity. Once anthracene enters the body, it appears to target the skin, stomach, intestines and the lymphatic system, and it is a probable inducer of tumors (Das et al., 2008).

Naphthalene binds covalently to molecules in liver, kidney and lung tissues, thereby enhancing its toxicity; it is also an inhibitor of mitochondrial respiration (Falahatpisheh et al., (2001). Acute

15 Literature Review naphthalene in humans can lead to haemolytic anaemia and nephrotoxicity. In addition, dermal and ophthalmological changes have been observed in workers occupationally exposed to naphthalene. Phenanthrene is known to be a photosensitizer of human skin, a mild allergen and mutagenic to bacterial systems under specific conditions (Mastrangela et al., 1996).

The degree of toxicity is generally related to the molecular weight of the PAH, with the higher molecular weight compounds often exhibiting greater toxicity. Carcinogenicity is particularly associated with high molecular weight PAHs such as benzo[a] pyrene. Mutagenicity studies have indicated that the only PAHs that are clearly not mutagenic are naphthalene, fluorene, and anthracene (Bamford and Singleton, 2005).

Toxicological studies have shown associations between exposure of animals to PAH and reproductive toxicity, cardiovascular toxicity, bone marrow toxicity, immune system suppression, liver toxicity and cancer (Collins et al., 1998). Epidemiological studies have shown evidence that cancer, birth defects, genetic damage (IARC, 2009), immunodeficiency (USEPA, 2007), respiratory (Andersson et al., 1997) and nervous system disorders (USEPA, 2007) can be linked to exposure to occupational levels of PAHs.

Moreover, exposure to genotoxic carcinogenic compounds at a young age may represent a health risk, i.e. by causing genetic damage (mutation, sister chromatid exchanges and other genetic disruption) (Neri et al., 2003).

VOC and/or PAH coated onto particulate matter (PM) induced gene expression of (cyp) 1a1, cyp2e1, nadph quinone oxydo-reductase-1, and glutathione S--pi 1 and mu 3, versus controls, suggesting thereby the formation of biologically reactive metabolites (Saint-Georges et al., 2008).

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Singh et al., (2007) proved that Polycyclic aromatic hydrocarbons (PAHs) appear to be significant contributors to the genotoxicity and carcinogenicity of air pollution present in the urban environment for humans. B[a]P, alone or in binary mixture with other PAHs, led to low amounts of strand breaks. Several biological mechanisms may account, including binding of PAHs to the Ah receptor (AhR), their affinity toward CYP450 and competition for (Tarantini et al., 2011).

The recent revival of interest in the study of mitochondria has been stimulated by the evidence that genetic and/or metabolic alterations in this organelle lead to a variety of human diseases including cancer (Priya et al., 2011).

Marston et al., (2001) indicated that trace amounts of PAHs can cause cancer, hypoplasia, and hypersensitivity responses in humans and elicits DNA damage, mutagenesis and carcinogenesis (Ling et al., 2004).

Human genomic DNA is continually subject to damage caused by exogenous and endogenous genotoxic agents. The progress of the replicative DNA polymerases (Pols), can be blocked by DNA lesions, possibly leading to cell death (Nohmi, 2006), double strand breaks (DSB) (Shrivastav et al., 2008).

IARC classify benzo(a)pyrene (BaP) as a known human and dibenz(a,h)anthracene (DahA) as probably carcinogenic to humans (IARC, 2009).

Both the World Health Organization and the UK Expert Panel on Air Quality Standards (EPAQS) have considered benzo(a)pyrene (BaP) as a marker of the carcinogenic potency of the polycyclic aromatic hydrocarbons (PAH) mixture (Delgado-Saborit et al., 2011).

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1.2.2. On animal:

Chemical pollution by petroleum hydrocarbons has a negative effect on natural marine ecosystems. Changes in the diversity, abundance, and activity of autochthonous populations, as a consequence of oil spills have been reported (MacNaughton et al., 1999; Megharaj et al., 2000; Saul et al., 2005; Bode et al., 2006).

Because of their importance in aquatic ecosystems and in human food sources, are frequently used as the standardized testing protocols for such purposes as predicting the bioconcentration factor (BCF) (Barron, 1990).

PAHs with molecular weight 278 have been found to be mutagenic or carcinogenic to a various degree. However, data on their carcinogenicity are mostly available only for dibenz(a,h)anthracene (DBahA), which is classified as probable human carcinogen (Švihálková- Šindlerová et al., 2007). The carcinogenic effects of these PAHs are mostly attributed to their ability to be metabolised to , forming DNA adducts in vitro (Giles et al., 1997). The dihydrodiol epoxides of BgChry and BcChry induce mammary tumours in female (Amin et al., 2003) and this metabolite of BgChry also induces high numbers of liver and lung tumours in newborn mice (Amin et al., 1995). Metabolites of DBacA and DBajA are mutagens, which may initiate tumours in mouse skin (Sawyer et al., 1988). Most of the carcinogenicity data have been obtained using either skin or newborn mice models. However, it is known that these compounds may not be carcinogenic in other models (Sellakumar and Shubik, 1974), and they are often only weak mutagens or genotoxins (Cheung et al., 1993). The carcinogenic effects of PAHs have been related mainly to their capacity to form electrophilic metabolites capable of covalent reaction with DNA to form DNA adducts (Baird et al., 2005). This suggests that they (PAHs) may

18 Literature Review act as tumour promoters, either directly or through formation of further active metabolites (Burdick et al., 2003).

The results clearly indicated that phenanthrene could induce .OH generation and result in oxidative stress in liver of (Yin et al., 2007).

Shi et al. (2005b) showed that naphthalene could induce reactive oxygen species (ROS) generation and result in oxidative damage in liver of Carassius auratus, and when exposed to pyrene (Sun et al., 2008).

Exposure to PAHs increases the expression of cytochrome P450 and reactive oxygen species (ROS) may be produced and oxidative stress (Meyer et al., 2002).

Wang et al. (2006) reported that benzo[a]pyrene affected the activities of hepatic antioxidant defense of Sebastiscus maramoratus.

There is a wide range of studies which indicate that fish embryos and larvae are highly sensitive to PAHs (Sundberg et al., 2005). Gross malformations resulting from exposure to PAHs include pericardial and yolk sac edema, jaw reductions, presumptive skeletal defects described as spinal malformations such as lordosis or scoliosis (dorsal curvature), and craniofacial skeleton disorders. Reductions in larval heart rate (bradycardia) and cardiac arrhythmia have also been observed (Incardona et al., 2009). Increased weathering of crude oil, which shifts the composition from predominantly two-ring (e.g., ) to three ring PAHs (e.g., Phe), result in a greater toxic potency and a higher frequency of malformations (Carls et al., 1999). Tricyclic AHs and weathered crude oil cause early cardiac dysfunction during key stages of cardiac morphogenesis. (Incardona et al., 2005). Another study indicates that some tetracyclic PAHs (pyrene and benz[a]anthracene) produce developmental toxicity through the AHR pathway (Incardona et al., 2006). In a recent study, severe histopathological alterations were

19 Literature Review observed in gonads of female Psammechinus miliaris following exposure to phenanthrene (Schäfer and Köhler, 2009).

Fibrosis involves augmentation of collagen and more massive appearance of the connective tissue (Dietrich et al., 2009), and phenanthrene exposure increases fibrosis in sea urchin gonads (Schäfer and Köhler, 2009). One of the key responses of tissues or cells to exposure is a decrease of available energy and reducing power due to energy dependent protective mechanisms against xenobiotics and chemical detoxification (Ataullakhanov and Vitvitsky, 2002).

Histopathology and reproduction in fish, mortality in fish embryos, changes in community structures, etc. were reported even years after the spill (Peterson et al., 2003).

The residual levels of PAHs in the liver, brain, gill and muscle tissues of four common edible freshwater fish species including crucian carp, snakehead fish, grass carp and silver carp collected from Small Bai-Yang-Dian in northern China were measured by GC–MS. Low molecular weight (LMW) PAHs predominated the distribution in the fish tissues, accounting for 89.97% of total PAHs (Xu et al., 2011).

The results from our previous assays performed with S. senegalensis showed that even moderate contamination levels have the potential of causing severe chronic lesions and alterations to fish (Chapman et al., 2002).

Wolfe et al., (2001) observed that the breakdown products, following the application of dispersants to crude oil, resulted in increased toxicity to eggs and early stages of a fish, top smelt (Atherinops affinis). Moreover, Ramachandran et al., (2004) observed that an oil dispersant increased PAH uptake in fish exposed to crude oil as induction of the detoxification CYP1A increased by 6 to 1100 fold than water-accommodated fractions (WAF) alone.

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The exposure of larvae to BaP, pyr. or phen. Na/K-ATPase and

Ca2 - ATPase activity suggesting that the developmental defects caused by PAHs were related to their inhibition of Na /K -ATPase and Ca2 -ATPase activity (Li et al., 2011).

The cyp1a gene proved to be the most sensitive and robust marker for oil contamination. In liver samples of fish, collected from different in the Urucu oil mining area, no elevated expression of cyp1a transcripts was observed (Matsuo et al., 2006 and dos Anjos et al., 2011).

Different responses and sensitivity to environmental toxicity between the two types of assays with fish and aquatic invertebrates have already been reported and some authors argued that both are important for biomonitoring purposes (Smolders et al., 2004), including those using flatfish as test organisms , a group of benthic vertebrates recognized as very sensitive to sediment-bound contamination (Johnson et al., 1998). The two biomarkers, DNA strand breakage and chromosome clastogenesis; reflect different types of DNA damage. The SCGE assay quantitates DNA fragmentation (Costa et al., 2011), single- or double-strand, through the neutral or alkaline versions, respectively, a type of damage that may result from direct DNA chain oxidation, formation of xenobiotic - DNA adducts and alkali-labile sites. This sort of mutagenesis depends on the action of the cellular DNA-repairing machinery (since single-strand damage may be reversible), (Sarasin, 2003).

It is now well appreciated that female and male fish of the same species can differ with respect to their susceptibility to environmental contaminants (Vega-Lopez et al., 2007 and Schäfer et al., 2011). Examples are seen in female flounder (Platichthys flesus L.), where higher incidence of is associated with sex-specific differences in NADPH metabolism important for xenobiotic biotransformation (Köhler and Van Noorden, 2003). Interestingly, in invertebrates male gametes

21 Literature Review often appear more susceptible to chemical stress than eggs (Fitzpatrick et al., 2008).

Fish were found to be more sensitive to the toxic effect of heavy (HFO) than were higher plants (Kazlauskienė et al., 2004).

Exposure of precision-cut liver slices to six structurally diverse polycyclic aromatic hydrocarbons, namely benzo[a] pyrene, benzo[b] fluoranthene, dibenzo [a,h] anthracene, dibenzo[a,l]pyrene, fluoranthene and 1-methylphenan-threne , led to induction of ethoxyresorufin O-deethylase, CYP1A apoprotein and CYP1A1 mRNA levels, but to a markedly different extent (Pushparajah et al., 2008).

The formation rate of DNA adducts has been shown to be orders of magnitude greater in the skin of mice exposed to B[a]P compared to internal organs, indicating the risk of skin tumors may exceed internal tumors following dermal exposure (Talaska et al., 1996).

1.2.3. On plant:

The volatile PAH compounds of Phenanthrene (PHE) and fluoranthene (FLU)) with a vapor phase component in the air are subject to an air-leaf exchange process moving towards equilibrium over time ( Wild et al., 2004).

Plants are very sensitive and respond rapidly to the presence of Phenanthrene (PHE) and fluoranthene (FLU) (Kummerová et al., 2006a,b).

PAHs exhibit a potential for bioaccumulation and have a negative effect on algae (Chan et al., 2006).

PAHs deposited on the surface of pine needles may induce the generation of reactive oxygen species in the photosynthetic apparatus, a

22 Literature Review manner closely resembling the action of the (Oguntimehin et al., 2007).

The effect of a hydrocarbon mixture (HCM) of three polycyclic aromatic hydrocarbons (PAH) and Maya crude oil on germination, growth and survival of four grasses was studied and compared to a control under in vitro conditions. Germination was not affected for any assayed concentration; however, the length of the stems and roots decreased when HCM increased and the survival of the four species also diminished (Reynoso-Cuevas et al., 2008).

Diatoms were exposed to polycyclic aromatic hydrocarbons mixture (PAH) from surface sediments collected at a highly PAH contaminated area of the Mediterranean Sea (Genoa, Italy), due to intense industrial and harbor activities (Carvalho et al., 2011).

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1.3. Petroleum oil contamination:

Contamination of soil and ground water by the accidental release of petroleum hydrocarbons (PHC) is a common problem for drinking water supplies (U.S. National Research Council 1993).

Soils surrounding crude-oil , fuel storage depots, and are some of the more common sites where industrial scale PAH pollution has been detected (Mahro et al., 1994). The zone where PHC are found as a free phase is designated as the source area (ASTM, 1995). Boulding (1995) revealed that at petroleum spill sites, PHC usually migrate vertically downward through the unsaturated zone due to the force of gravity and then laterally along the ground water table.

Marine pollution due to oil spills is one of the most prevalent environmental and economic concerns worldwide (Readman et al., 1992; Fowler et al., 1993; Sauer et al., 1998).

Crustacean heart rate is a useful biomarker. Cardiac activity increased in Carcinus maenas following exposure to the water soluble fraction of crude oil (Depledge, 1984).

Oil spills can have severe consequences for the ecological equilibrium of aquatic ecosystems. This has been observed particularly for marine environments with the Exxon Valdez, Amoco Cadiz and Prestige oil spills as some of the most prominent examples. In these spills, direct physical contact and/or exposure to toxic compounds released from oil caused high mortality rates in algae, invertebrates, sea mammals and birds (Peterson et al., 2003).

Daane et al., (2001) found that the Iraqi forces deliberately damaged more than 700 Kuwaiti oil wells. Oil remained gushing for about

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7 months resulting in the heavy contamination of about 50 km2 of the desert area.

Oil pollution from several large-scale oil fields in the northeast of China remains one of the major environmental problems in these areas (Stanford et al., 2007).

1-4. Polycyclic aromatic hydrocarbons as constituent of petroleum contaminants:

Leahy and Colwell (1990) revealed that petroleum is a complex mixture of hydrocarbons and related compounds generally classified in four fractions: aliphatics, aromatics, polars or and . Aromatic and polar constituents are less biodegradable than aliphatics, while asphaltenes are regarded as nonbiodegradable.

Aromatic hydrocarbons are common pollutants found in soil and groundwater as a result of past and current industrial activity (Mueller et al., 1996).

Some PAHs (e.g. naphthalene and phenanthrene) have also been used in the synthesis of different organic compounds in pesticides, fungicides, detergents, dyes and mothballs (Shennan, 1984).

Environmental pollution caused by the release of crude and refined petroleum products occurs in all areas of the world. These complex may consist of hundreds of compounds that are highly variable in structure and in susceptibility to biodegradation (Bossert and Compeau, 1995).

Petroleum components have traditionally been divided into four fractions: saturated hydrocarbons, aromatic hydrocarbons, -sulphur- oxygen containing compounds (NSO) and asphalthenes. The relative proportions of these fractions vary from crude to crude, and the susceptibility

25 Literature Review of a specific crude to microbial degradation can be predicted from its composition. Normally, the fractions contain n- and are mostly susceptible to biodegradation, whereas saturated fractions containing branched alkanes are less vulnerable to microbial attack. The aromatic fractions are even less easily biodegraded, and the susceptibility decreases as the number of aromatic or alicyclic rings in the increases (Barthakur, 1997).

Petroleum is a complex mixture of hydrocarbons and other organic compounds including organometallic complexes of and vanadium (van Hamme et al., 2003).

The oil contains , particularly , and, in lesser quantities, nickel, aluminum, and vanadium, in addition to sulfur and polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOCs) such as benzene, , , and (Bosch, 2003).

Polycyclic aromatic hydrocarbons (PAHs) are widespread contaminants. They are produced during the incomplete combustion of coal, gas, oil, and wood (National Research Council, 2003; Douben, 2003 and Banford and Singleton, 2005).

Casablanca crude oil is aliphatic with a low ; Maya represents a sulphur-rich that is predominantly aromatic (Llirós et al., 2007).

Mancera-López et al., (2008) found that a complex mixture of total petroleum hydrocarbons (TPH), which comprises 40% aliphatic hydrocarbons (AH) and 21% polycyclic aromatic hydrocarbons (PAH).

Hong et al., (2008) indicated that polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants generated

26 Literature Review

from both natural and anthropogenic processes. Therefore, they pose serious threats to the health of aquatic and human life through bioaccumulation.

Polycyclic aromatic hydrocarbons (PAHs) may be present in high concentrations at industrial sites associated with the petroleum, coal-tar, gas production and wood preservation industries (Xu et al., 2006 and Somtrakoon et al., 2008). As toxic, mutagenic and carcinogenic chemicals that are ubiquitous in environment (Yucheng et al., 2008 and Hong et al., 2008).

Polycyclic aromatic compounds, (PACs) are a diverse group of hydrophobic organic contaminants that enter the aquatic and terrestrial environments from multiple sources, including the production, transport, and combustion of fossil fuels (Mastral and Callén, 2000; Lima et al., 2005; Wang et al., 2006 and Callén et al.,2011).

The environmental contamination with petroleum hydrocarbons due to industrial wastes and oil spill accidents is widespread. Diesel oil is one of the major contaminants of soil and groundwater (Johnsen et al., 2005 and Tang et al., 2005 and Márquez-Rocha et al., 2005).

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants persisting in the environment. Anthropogenic inputs of PAHs from oil spills, ship traffic, urban runoff and emission from combustion and industrial processes have caused significant accumulation of PAHs in coastal environments (Volkering et al., 1992; Mueller et al., 1996; Hughes et al., 1997 and Xu et al., 2005).

They are natural components of fossil fuels such as petroleum and coal, and may enter the environment as a result of accidental spills and natural leakage of these products. PAHs are also formed during the incomplete combustion of organic matter during volcanic activity, forest and prairie fires, combustion, waste incineration, and to a lesser

27 Literature Review extent, the cooking of food (Maliszewska-Kordybach, 1999). Other significant sources of PAHs include and creosote, both by-products of coke production. Coal tar residue is a by-product of coal and up to 300 g kg_1 total PAHs have been reported in soils at abandoned sites (Sutherland et al., 1995). Creosote, a high distillate of coal tar, has been used for over a century in the wood preservation industry, and consists of a complex mixture of organic chemicals including (5%), N-, S-, and O-heterocyclics (10%), and polycyclic aromatic hydrocarbons (85%) (Rasmussen and Olsen, 2004).

The primary sources of PAHs are mainly from the incomplete combustion of various organic matters such as fossil fuels (e.g. coal, and diesel) and biomass fuels (e.g. straw, firewood) (Jacques et al., 2008 and Zhang and Tao, 2009). In many developed counties, the PAHs emissions have significantly decreased because of the improved efficiency of energy utilization in the past decades (Sun et al., 2006). However, in China, the PAH emissions have been increasing greatly due to the increasing energy demand associated with rapid population growth and economic development, and to the low efficiency of energy utilization (Zhang et al., 2007).

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that enter the environment via incomplete combustion of fossil fuels and accidental leakage of petroleum products, and as components of products such as creosote (Haritash and Kaushik, 2009 and Muckian et al., 2009).

Polycyclic aromatic hydrocarbons (PAH) are the main components of emissions generated by coke oven factories and many of these chemicals are carcinogenic (Castorena-Torres et al., 2008 and Perrello et al., 2009).

Among man-made substances that cause ecotoxicological problems are a variety of aromatic compounds such as halogenated aromatic 28 Literature Review compounds, polycyclic aromatic hydrocarbons (PAHs) The main sources of these toxic substances are oil refineries, gas-stations, use of wood and agro -chemicals, and pharmaceutical industries (Madsen, 1991 and Budavari, 1996).

Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of organic compounds containing two or more fused aromatic rings derived from the incomplete combustion of organic matters including coal, oil, gas, wood, garbage, or other organic substances, such as tobacco and charbroiled (Bamford and Singleton, 2005; Jacques et al., 2008 and Rey-Salgueiro et al., 2008).

Polycyclic aromatic hydrocarbons (PAHs) represent a large and diverse group of organic molecules having a broad range of properties, differing in molecular weight, structural configuration, water solubility, number of aromatic rings, , sorption coefficients, etc. (Venkata Mohan et al., 2006).

PAH are difficult to remove from soil due to their recalcitrant nature and, apart from naphthalene, they are practically insoluble in water and slow to degrade (Kottler and Alexander, 2001). Their persistence in the environment is related to their low aqueous solubility, vapor and high octanol/water partitioning coefficients (Oleszczuk and Baran, 2003 and Haritash and Kaushik, 2009). As a consequence, PAHs have a high affinity for association with organic carbon material (humus) in soil (MacGillivray and Shiaris, 1994; Alexander, 1995; Juhasz and Naidu, 2000 and Jonsson et al., 2007).

PAHs are usually bound to suspended particles in aquatic ecosystems and ultimately deposited into sediments (Cerniglia and Heitkamp 1989; Hughes et al., 1997 and Tam et al., 2001).

The group of polycyclic aromatic hydrocarbons (PAHs) comprises over 100 diverse compounds that are characterized by fused ring systems

29 Literature Review and can be detected in soils, sediments and groundwater (Doyle et al., 2008).

PAHs are a diverse group of over 100 organic compounds containing two or more fused benzene and/or pentacyclic ring structures, in linear, angular, or cluster arrangements .They are thermodynamically stable due to their negative energy and possess high melting and boiling points together with low water and vapor pressures (WHO, 1998).

Polycyclic aromatic and heavier aliphatic hydrocarbons, which have a stable recalcitrant molecular structure, exhibit high hydrophobicity and low aqueous solubility, are not readily removed from soil through leaching and volatilization (Brassington et al., 2007).

The hydrophobicity of PAHs limits desorption to the aqueous phase (Donlon et al., 2002).

1-4-1. Number of rings in PAHs compounds:

Polycyclic aromatic hydrocarbons (PAHs) require serious consideration among the toxic pollutants because of their ubiquitous distribution, environmental persistence and potentially deleterious effects on human health. PAHs represent a large and diverse group of organic molecules having a broad range of properties, differing in molecular weight, structural configuration, water solubility, number of aromatic rings, volatility, sorption coefficients, etc. (Harmsen, 2004 and Giordano et al., 2005).

Juhasz and Naidu (2000) showed that in natural environments, the low molecular weight (LMW) PAHs (consisting of 2–3 aromatic rings) are relatively easy to be degraded, while the high molecular weight (HMW) PAHs (containing 4 or more aromatic rings) are persistent .

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2 rings (naphthalene):

Naphthalene (Naph.), the first member of PAH group and one of the 16 PAHs classified as priority pollutants by Environmental Protection Agency (EPA) of United States, is a frequent pollutant established in nature. This bicyclic aromatic hydrocarbon and its methylated derivatives are considered some of the most noxious compounds in the water-soluble fraction of petroleum (Heitkamp et al., 1987).

Studies of naphthalene degradation may be significant because naphthalene is a common pollutant that serves as a chemical model for the degradation of PAH (Ahn et al., 1999).

Naphthalene, the first member of the PAH group, is a common micropollutant in potable water. The toxicity of naphthalene has been well documented (Goldman et al., 2001).

3 ring-*Phenanthrene:- Among other PAHs, phenanthrene (Phen.), a polycyclic aromatic hydrocarbon with three condensed rings fused in angular fashion is widely distributed in the environment primarily because of anthropogenic and pyrolytic processes. Phenanthrene has been used in the synthesis of different organic compounds like pesticides, fungicides, detergents, dyes and mothballs (Shennan, 1984).

Phenanthrene is known to be a photosensitizer of human skin, a mild allergen and mutagenic to bacterial systems under specific conditions (Fawell and Hunt, 1988).

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Phenanthrene, a tricyclic PAH, which is considered the most abundant hydrocarbons in the aquatic environment (Chen et al., 2004) was chosen as a test compound.

Moreover, several countries set phenanthrene ‘safe’ levels for aquatic organisms equal or less than 4.6 mg L_1 (Law et al., 1997).

3-ring-Anthracene:- Anthracene (Anth.) is a non-mutagenic and non-carcinogenic, low-molecular-weight polycyclic aromatic hydrocarbon present in the environment. Its toxicity can be dramatically increased after solar-light exposure (Guiraud et al., 2008).

3-ring-hetero-Fluorene Fluorene (Flu.), a tricyclic PAH with two benzene rings fused to a ring, is formed during the combustion of fossil fuels, such as in the oil process, and in automotive tailpipes. This xenobiotic compound and its derivatives are a major environmental concern associated with petroleum and oil spills, waste incineration, and industrial effluents (Lu and Zhu, 2007). They are insoluble in water but soluble in organic . Fluorene and its constituents have many applications in industry, since they are used as base materials for dyes and optical brightening agents. In addition, some of their characteristics, such as light and temperature sensitivities, heat resistance, conductivity, and resistance, make them applicable for use in the areas of thermo and light sensitizers, luminescence chemistry, spectrophotometric analysis, and molecular chemistry (Yuanfu et al., 2007).

4-ring-Pyrene:-

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The International Agency for Research on Cancer (IARC) classifies pyrene as a group 3 carcinogen (unclassifiable as a human carcinogen). Although being noncarcinogenic and not phototoxic/toxic to freshwater green alga, Selenastrum capricornutum (Warshawsky et al., 1995), pyrene was found acutely toxic to marine alga Phaeodactylum tricornutum (Okay et al., 2002).

Pyrene, a four-ring PAH that has a low biodegradability and high persistence in the environment, is one of the PAHs on the United States Environmental Protection Agency (US EPA) priority pollutant list (Yan et al., 2004).

4-ring-Fluoranthene (hetero) Fluoranthene (Fluo.) and pyrene were selected because they were the main representative PAHs, predominant in air, sediment and water, and were often existed together in contaminated environments (Tang et al., 2005). They have the same molecular weight , but the solubility of fluoranthene (0.26mg l-1) was nearly double of that of pyrene (0.14mg l-1) (Mackay et al., 1992).

4-ring benzo(a)anthracene Benzo(a) anthracene (BaA) was chosen as the model PAH compound due to its common presence in several matrices in the marine environment.

5-ring Benzo-a- pyrene Benzo(a)pyrene (BaP) and related polycyclic aromatic hydrocarbons (PAHs) were present in broiled beef. Since then, studies have provided much information on the levels of carcinogens found in grilled meat products (Sundararajan et al., 1999).

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Benzo[a]pyrene (BaP), a fused pentacyclic aromatic hydrocarbon, has a very low aqueous solubility (3.8 g/L) and a high octanol/water partitioning coefficient (6.04), which suggests its preference for non- aqueous phases (Rivas, 2006). It has been classified by the US Environmental Protection Agency as a priority pollutant because of its carcinogenicity, teratogenicity and acute toxicity. Such polycyclic aromatic hydrocarbons (PAHs) are quite recalcitrant to biodegradation in soil because of their aromatic and condensed structure leading to low solubility and then high chemical stability.

In locations strongly influenced by traffic, this source (BaP), can provide 88% of the total of this compound (Lee and Jones, 1999). B[a]P is a polycyclic aromatic hydrocarbon (PAH) that is lipophilic, of low solubility in water, and has a high affinity for organic matter in soil (United States Environmental Protection Agency, 2002).

Emphasis has been placed on the genotoxic properties of benzo[a]pyrene (B[a]P) the only one PAH classified as carcinogenic to humans by the International Agency of Research on Cancer (IARC) (IARC, 2010).

1.5. Microorganisms degrading PAHs.

Six main ways of dissipation, i.e. disappearance, are recognized in the environment: volatilization, photooxidation, chemical oxidation, sorption, leaching and biodegradation. Microbial degradation is considered to be the main process involved in the dissipation of PAH (Cerniglia, 1992; Sutherland et al., 1995 and Yuan et al., 2002).

The fate of PAHs and other organic contaminants in the environment is associated with both abiotic and biotic processes, including volatilisation, photooxidation, chemical oxidation, bioaccumulation and microbial transformation (Boonchan et al., 2000).

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Compared with other physical and chemical methods such as combustion, photolysis, landfill and ultrasonic decomposition, biodegradation is expected to be an economic and environmentally friendly alternative for removal of PAHs (Giraud et al., 2001; Schloter et al., 2003 and Toledo et al., 2006). Thus, more and more research interests are turning to the biodegradation of PAHs. Some microorganisms can utilize PAHs as a source of carbon and energy so that PAHs can be degraded to carbon dioxide and water, or transformed to other nontoxic or low-toxic substances (Viñas et al., 2002; Medina-Bellver et al., 2005; Fernández-Álvarez et al., 2006; Gallego et al., 2006; Jiménez et al., 2006; Gallego et al., 2007; Santos et al., 2008 and Perelo, 2010).

The degradation of petroleum-derived hydrocarbons has been widely studied and it has been established that microbial degradation is a key removal pathway of hydrocarbons from the soil matrix (Bogan et al., 2003).

Tian et al., (2002) indicated that microbial degradation of PAHs is considered to be the major decomposition process for these contaminants in nature, and represents a potential solution to the environmental problems posed by them.

Microorganisms play a major role in the biogeochemical cycling of both organic and inorganic elements, as they are the main mediators of biodegradation and mineralization of organic compounds (Madigan et al., 2000).

Pan et al. ,(2004) indicated that a wide varieties of resources have been found to be available for PAH degradation.

Márquez-Rocha et al., (2005) revealed that many isolated bacterial and fungal species have been reported to be capable of biodegrading effectively petroleum hydrocarbons and even polynuclear aromatic hydrocarbons.

35 Literature Review

The ability of bacteria to degrade polycyclic (PACs) depends primarily on PAC bioavailability, which decreases with increasing numbers of aromatic rings and the degree of (Johnsen et al., 2005).

1.5.1. Bacteria degrading PAHs:

Mycobacterium spp., Sphingomonas spp., spp., and Nocardia spp. populations were selectively stimulated in soil contaminated with PAH or . Research on the of PAH-contaminated sites has frequently led to the isolation of degradative strains of fast-growing species of mycobacteria (Lloyd-Jones and Hunter, 1997).

A large number of bacteria that metabolize PAHs have been isolated (Alcaligenes denitrificans, Rhodococcus sp., Pseudomonas sp., Mycobacterium sp.) (Harayama, 1997). A variety of bacteria can degrade certain PAHs completely to CO2 and metabolic intermediates (Müncnerová and Augustin, 1994).

A large number of naphthalene-degrading microorganisms (including Alcaligenes denitrificans, Mycobacterium sp., Pseudomonas putida, P. fluorescens, P. paucimobilis, P. vesicularis, P. cepacia, P. testosteroni, Rhodococcus sp., Corynebacterium venale, Bacillus cereus, Moraxella sp., Streptomyces sp., Vibrio sp. and Cyclotrophicus sp.) has been isolated and examined for mineralization (Samanta et al. 2001). Efficiency phenanthrene degradation by different bacteria including Aeromonas sp., Alcaligenes faecalis, A. denitrificans, Arthrobacter polychromogenes, Beijerinckia sp., Micrococcus sp., Mycobacterium sp.,Pseudomonas putida, P. paucimobilis, Rhodococcus sp., Vibrio sp., Nocardia sp., Flavobacterium sp., Streptomyces sp. and Bacillus sp. (Samanta et al., 1999).

The degrading strains that have been characterized so far in the literature are taxonomically diverse, and mainly belong to the genera

36 Literature Review

Pseudomonas, Alcaligenes, Sphingomonas, Bacillus and Mycobacterium (Aitken et al., 1998; Baldwin et al., 2000; Barathi and Vasudevan, 2001 and Cheung and Kinkle, 2001).

Using bioremediation technology to cleanup PAH-contaminated sites has been suggested to be an efficient, economical and versatile alternative to physicochemical treatment (Margesin and Schinner, 1997). Nowadays, in order to eliminate PAHs from environment by bioremediation, many PAHs- degradation microorganisms were isolated. Most of these microorganisms belong to Pseudomonas sp. (El-Nass et al., 2009). Mycobacterium (Pagnout et al., 2007). Rhodococcus (Martínkov et al., 2009), Neptunomonas (Li and Chen, 2009)., Stenotrophomonas (Liang et al., 2008), Cycloclasticus (Kasai et al., 2002), Staphylococcus (Mallick and Dutta, 2008), Burkholderia (Tillmann et al., 2005), Acinetobacter, Agmenellum, Aeromonas, Bacillus, Berjerinckia, Corynebacterium, Flavobacterium, Micrococcus, Moraxella, Nocardioides, Lutibacterium, Streptomyces, Vibrio, Paenibacillus and some fungi (Rafin et al., 2009).

PAH-degrading mycobacteria display various physiological adaptations for survival in PAH contaminated soils, such as high specific affinities for PAH (Uyttebroek et al., 2006) or formation on PAH-containing surfaces.

Mycobacterium, Sphingomonas, Terrabacter and Rhodococcus were isolated from a single surface sediment sample, all four genera could degrade three and four-ring PAHs, their in situ activities in natural sediment slurry were found to be different. A cultivable method showed that Sphingomonas strains grew rapidly under the induction of three-ring, but not four-ring while only Mycobacterium degrading strains dominated in the four-ring PAHs (Zhou et al., 2008).

Cébron and Norini (2008) found that the Gram negative PAH degraders such as Pseudomonas, Ralstonia, Commamonas, Burkholderia, Sphingomonas, Alcaligenes, Polaromonas strains, and the Gram positive 37 Literature Review

PAH degraders such as Rhodococcus, Mycobacterium, Nocardioides and Terrabacter strains .

Guo et al., (2010) indicated that most PAH-degraders microorganisms are in the Pseudomonas and Sphingomonas , but others are in the Alcaligenes, Mycobacterium, Rhodococcus, or Bacillus genera, and additionally are fungi.

It has been observed that PAH degradation in soil is dominated by bacterial strains belonging to a very limited number of taxonomic groups such as Sphingomonas, Burkholderia, Pseudomonas and Mycobacterium. Among these taxonomic groups a high proportion of the PAH- degrading isolates belong to the Sphingomonads sensulato (Johnsen et al., 2002).

Petroleum and polycyclic aromatic hydrocarbons (PAHs) degrading Streptomyces sp. isolate ERI-CPDA-1 was recovered from oil contaminated soil in Chennai, India. The degradation efficiencies were examined by GC-FID and the results showed that the isolate could remove 98.25% diesel oil, 99.14% naphthalene and 17.5% phenanthrene in 7 days at 30oC (0.1%) (Balachandran et al., 2012).

He-Ping et al. (2008) isolated and characterized two PAH- degrading bacteria from the polluted Chinese soils, including the first representative of strain Tistrella sp. ZP5 able to increase the speed of phenanthrene degradation when inoculated with another phenanthrene- degrading bacterium strain Sphingomonas sp. ZP1.

Alcanivorax and Cycloclasticus of the g- were identified as two key organisms with major roles in the degradation of petroleum hydrocarbons. is responsible for biodegradation, whereas Cycloclasticus degrades various aromatic hydrocarbons. This information will be useful to develop in situ bioremediation strategies for the clean-up of marine oil spills (Harayama et al., 2004).

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Many bacterial strains have been isolated from coastal and oceanic environments; these bacteria, including the genera Pseudomonas, Vibrio and Flavobacterium, have been considered to be representative of marine bacteria (Amann et al., 1995).

Habe and Omori (2003) indicated that the aerobic biodegradation process also known as aerobic respiration is the breakdown of contaminants by microorganisms in the presence of oxygen. Aerobic bacteria use oxygen as an electron acceptor to break down both the organic and inorganic matters into smaller compounds, often producing carbon dioxide and water as the final product.

Our hypothesis, based on recent bacterial research (Xu et al., 2004; Grabowski et al., 2005), goes further by suggesting that anaerobic bacteria present in water droplets might be trapped in bacterial at the water/oil interface, producing oxygen by reducing and perchlorates during anaerobic life cycles. The biofilm would therefore act as an oxygen sponge that could have either high or low oxygen content. Anaerobic biodegradation activity would then slowly decrease with increasing oxygen content and the degradation activity would be taken over by the aerobic consortium, consequently consuming the stored oxygen.

Due to the potential advantages of using elevated for bioremediation, several researchers have recently studied for degradation of hydrocarbons, including long chain alkanes, aromatics and PAH compounds. These organisms predominantly belong to the genera Thermus or Geobacillus (Feitkenhauer et al., 2003).

Toledo et al., (2006) observed that fifteen bacterial strains isolated from solid waste oil samples were selected due to their capacity of growing in the presence of hydrocarbons. The majority of the strains belonged to genera Bacillus, Bacillus pumilus and Bacillus subtilis.

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Besides, three strains were identified as Micrococcus luteus, one as Alcaligenes faecalis and one strain as Enterobacter sp.

Biotransformation capacities of Anthracene by Tetrahymena pyriformis and a selection of eight micromycetes were studied, and the ability of these microorganisms to detoxify the polluted ecosystems was assessed. We showed that T. pyriformis was able to accumulate high amounts of anthracene (AC) without any transformation (Guiraud et al., 2008).

Bacteria are considered to represent the predominant agents of hydrocarbon degradation in the environment (Leahy and Colwell, 1990), and hydrocarbon-degrading bacteria are ubiquitous. More than 20 genera of marine hydrocarbon-degrading bacteria, distributed over several (sub)phyla Proteobacteria; Gram positives; Flexibactercytophaga-Bacteroides have been described so far (Engelhardt et al., 2001). As a single species typically is capable of degrading only a limited number of the compounds found in crude oil, a consortium composed of many different bacterial species is usually involved in oil degradation.

Escherichia coli is known to respond to certain toxic chemicals through an increased expression of various stress genes, as a representative for DNA, oxidative, membrane and protein damage, after E. coli was exposed to different polycyclic aromatic hydrocarbons (PAHs), i.e., phenanthrene, naphthalene and benzo[a]pyrene. Tests with the PAHs showed naphthalene and benzo[a]pyrene to be genotoxic, while phenanthrene had no clear effect on the expression of any of these genes (Kim et al., 2007).

We postulated that the surface hydrophobicity of P. putida NCIB 9816-4 in the exponential growth phase might be increased during the uptake of naphthalene, which caused the preferred adhesion to the naphthalene-contaminated soil (Hwang et al., 2009).

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The microalgal species Skeletonema costatum and Nitzschia sp. were capable of accumulating and degrading the two typical PAHs simultaneously. The accumulation and degradation abilities of Nitzschia sp. were higher than those of S. costatum. Degradation of FLA by the two algal species was slower, indicating that FLA was a more recalcitrant PAH compound. The microalgal species also showed comparable or higher efficiency in the removal of the PHE–FLA mixture than PHE or FLA singly, suggesting that the presence of one PAH stimulated the degradation of the other (Hong et al., 2008).

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1.5.2. Fungi degrading M.O.

Prerequisites for successful biodegradation of organopollutants in soil are: (i) survival of the introduced fungi in the soil,(ii) active fungal growth through the soil or massive introduction of a pre-grown fungus into the whole volume of contaminated soil, and (iii) production and persistence of fungal enzymes (extracellular and intracellular) attacking the pollutant molecule(s) during growth in the soil. Another important factor known to influence the biodegradability of PAH molecules is their sorption to soil particles that depends on the content of soil organic carbon and can negatively affect bioavailability of PAHs and consequently reduce the efficiency of degradation (Shuttleworth and Cerniglia, 1995 and Steffen et al., 2007).

Nonligninolytic fungi generally oxidize PAHs via cytochrome P450 monoxygenases, resulting in the production of an arene that is subsequently hydrolyzed by an hydrolase to a trans-dihydro-diol. While many of these fungi can transform PAHs to transdihydrodiols and other oxidized products such as phenols, tetralones, , dihydrodiol epoxides, and various conjugates of the hydroxylated intermediates, few have the ability to mineralize PAHs (Mueller et al., 1996). Fungal metabolites produced are often more water-soluble and chemically reactive than the parent PAH, thus increasing their potential for mineralization by indigenous soil bacteria (Cerniglia, 1997). Ligninolytic fungi, such as chrysosporium and Trametes versicolor, use extracellular enzymes involved in degradation, such as lignin peroxidase, peroxidase, and other H2O2-producing peroxidases or , to degrade PAHs (Bezalel et al., 1997). These enzymes oxidize a wide range of organic compounds in a nonspecific radical-based reaction. This results in the production of quinines and , rather than dihydrodiols.

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Among degradation microorganisms, white-rot fungi have demonstrated the ability to degrade a wide range of pollutants, including PAHs (Haritash and Kaushik, 2009).

1-6. Mechanism of PAHs degradation strains:

Degradation by bacteria occurs primarily under aerobic conditions involving oxygenase-mediated ring oxidation and subsequent catabolite formation, ring fission and metabolism (Chang et al., 1996).

Sediments from the less heavily contaminated site that had been adapted for rapid anaerobic degradation of high concentrations of benzene did not oxidize naphthalene, suggesting that the benzene- and naphthalene-degrading populations were different (Coates et al., 1997).

The first step in the microbial degradation of PAHs is the action of dioxygenase, which incorporates of oxygen at two carbon atoms of a benzene ring of a PAH resulting in the formation of cis-dihydrodiol (Kanaly and Harayama, 2000). which undergoes rearomatization by to form dihydroxylated intermediates. Dihydroxylated intermediates subsequently undergo ring cleavage and form TCA-cycle intermediates (Sabate et al., 1999).

The biological degradation of PAHs can serve three different functions. (i) Assimilative biodegradation that yields carbon and energy for the degrading organism and goes along with the mineralization of the compound or part of it. (ii) Intracellular detoxification processes where the purpose is to make the PAHs water-soluble as a pre-requisite for of the compounds. Generally, it seems that intracellular oxidation and of PAHs in bacteria is an initial step preparing ring fission and carbon , whereas in fungi it is an initial step in detoxification (Cerniglia, 1984). (iii) Co-metabolism, which is the degradation of PAHs without generation of energy and carbon for the cell metabolism. Co-metabolism is defined as a non-specific enzymatic

43 Literature Review reaction, with a substrate competing with the structurally similar primary substrate for the enzyme’s active site. An example is the co- metabolization of benzo(a)pyrene by bacteria growing on pyrene (Boonchan et al., 2000). Keck et al. (1989) noted that: "In the case of a pure culture, co-metabolism is a dead-end transformation without benefit to the organism. In a mixed culture or in the environment, however, such an initial co-metabolic transformation may pave the way for subsequent attack by another organism".

To study the mechanism-based of PAHs in the E. coli, therefore, four stress responsive genes, were selected. Each gene responds to specifically to DNA damage, oxidative damage caused by hydroxyl radicals, membrane damage and protein damage (Peters et al., 2004).

Kotterman et al., (1998) demonstrated that the initial attack on HMW-PAHs in soil by fungal exoenzymes appears to be more likely than attack by bacterial intracellular enzymes. Fungal exoenzymes have the advantage that they may diffuse to the highly immobile HMW-PAHs. This is in contrast to bacterial PAH- dioxygenases, which are generally cell- bound because they require NADH as a co-factor. Oxidation products of PAHs are more soluble than the parent compounds and therefore more bioavailable to the microbial community.

The efficiency of PAH biodegradation in sediment was different from that in liquid medium. Some studies showed that PAH biodegradation was reduced by sorption to sediments (Ramirez et al., 2001) as highly lipophilic PAH tended to sorb tightly on sediments and limited its availability to microorganisms. However, other studies reported that sediments did not inhibit and even enhanced the biodegradation of PAHs (Laor et al., 1999).

Supaka et al., (2001) found that HMW PAHs may be biodegraded via using LMW PAHs or degradation pathway intermediates as carbon and energy source.

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Like many organic compounds, PAHs interact with cellular macromolecules only after metabolic activation. This process aims at converting the chemicals into water-soluble derivatives in order to facilitate their elimination. In the case of B[a]P, the two main phase I enzymes are the cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) (Shimada and Fuji-Kuriyama, 2004). The phase II enzymes involved in the subsequent detoxification step are mainly gluthatione-S-transferase (GST), UDP-glucurosyltransferase (UGT) and sulfotransferase (SULT). Induction of phase I and phase II enzymes is mediated by the aryl hydrocarbon receptor (AhR) signaling pathway that is activated by the binding of the contaminating molecule on AhR, a cytosolic protein (Denison et al., 2002). After binding, AhR translocates to the nucleus and binds to its partner, the Ah receptor nuclear translocator (Arnt). This heterodimer interacts with the xenobiotics response element sequence (XRE) present upstream in promoters of the genes of phase I and phase II enzymes leading to an up-regulation of the transcription of these genes and thus an increase in protein concentration (Denison and Nagy, 2003).

1.6.1. Role of in PAHs degradation:

Chemically and biologically produced are known to enhance the solubility of hydrophobic organic compounds like PAHs (Aronstein et al., 1991; Tiehm et al., 1997; Margesin and Schinner, 1999).

Banat (1995) revealed that surfactants can increase mobility and surface area available for microbial cell contact with hydrocarbons.

A possible way of enhancing the bioavailability of hydrophobic organic compounds is the application of (bio) surfactants, molecules which consist of a hydrophilic part and a hydrophobic part. Because of this property these molecules tend to concentrate at surfaces and interfaces and to decrease levels of and interfacial tension. Another

45 Literature Review important characteristic of surfactants is the fact that above a certain concentration (the critical micelle concentration) aggregates of 10 to 200 molecules, which are called micelles, are formed. The effect of a surfactant on the bioavailability of organic compounds can be explained by three main mechanisms: (i) dispersion of nonaqueous-phase liquid hydrocarbons, leading to an increase in contact area, which is caused by a reduction in the interfacial tension between the aqueous phase and the nonaqueous phase; (ii) increased solubility of the pollutant, caused by the presence of micelles which may contain high concentrations of hydrophobic organic compounds, a mechanism which has been studied extensively previously (Edwards et al., 1992); and (iii) ‘‘facilitated transport’’ of the pollutant from the solid phase to the aqueous phase, which can be caused by a number of phenomena, such as lowering of the surface tension of the pore water in soil particles, interaction of the surfactant with solid interfaces, and interaction of the pollutant with single surfactant molecules.

Laboratory studies have demonstrated that some combinations of non-ionic surfactants and bacterial strains, pure cultures as well as consortia, stimulate the biodegradation of PAH in liquid or soil systems (Lantz et al., 1995). However, in other cases the presence of surfactants resulted in no or decreased PAH degradation (Liu et al., 1995). Surfactants, if applied above the critical micelle concentration (CMC), have been reported to be toxic toward microorganisms in soil/water systems (Laha and Luthy, 1992). These observations can be explained by one or more of the following effects: (a) toxicity of surfactants due to surfactant-induced permeabilisation or lysis of the bacterial cell membrane (Heipieper et al., 1994); (b) toxicity of surfactant-enhanced aqueous PAH concentrations; (c) physical-chemical effects resulting in undesirable bacterial-cell/surfactant interactions, e.g., prevention of bacterial adhesion to the hydrophobic substrate (Neu, 1996). Competitive substrate utilization (Liu et al., 1995).

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Willumsen and Karlson (1998) found that surfactants are known to increase the apparent aqueous solubility of polycyclic aromatic hydrocarbons (PAHs) and may thus be used to enhance the bioavailability and thereby to stimulate the biodegradation of these hydrophobic compounds. However, surfactants may in some cases reduce or inhibit biodegradation because of toxicity to the bacteria.

Surfactants applied at concentrations above the critical micelle concentration (CMC) are known to enhance the mobility and apparent solubility of hydrophobic compounds (Edward et al., 1991). Therefore, use of surfactants has been proposed as a mechanism to enhance the bioavailability of hydrophobic pollutants for microbial degradation (Thiem 1994). Amendment with the non-ionic surfactant, X-100, has previously been shown to stimulate the mineralization of fluoranthene by Sphingomonas paucimobilis when grown in Bushnell-Haas medium (Lantz et al., 1995).

Biologically produced surfactants have less toxicity to microorganisms and may not sequester the hydrocarbons too strongly (Siñeriz et al., 2001).

The presence of the synthetic surfactants resulted not only in increased apparent solubilities but also in increased maximal rates of dissolution of crystalline naphthalene and phenanthrene. In activity and growth experiments, no toxic effects of the surfactants at concentrations up to 10 g /1 were observed. (Volkering et al., 1995).

Surface-active compounds may increase the bioavailability, either by increasing apparent hydrocarbon solubility in the aqueous system or by increasing the contact surface by means of stable emulsions (Rosenberg and Ron 1999). In most cases, the addition of synthetic surfactants inhibits biodegradation (Rouse et al., 1994) by being toxic to the microorganisms or strongly partitioning the hydrocarbons. On the other

47 Literature Review hand, biologically produced surfactants (i.e., biosurfactants) do not have harmful effects on the environment, are not toxic to microorganisms, and may not sequester the hydrocarbons too strongly (Desai and Banat, 1997).

It was observed that when nonionic surfactants were present at levels above critical micelle concentrations (CMCs), phenanthrene degradation was completely inhibited by the addition of Brij 30 and Brij 35, and delayed by the addition of Triton X100 and Triton N 101 (Yuan et al., 2000).

Furthermore, surfactants may on their own pose a risk to soil living species (Jensen et al., 2001).

Liu et al., (1995) indicated that experimental results showed that surfactant concentrations above the critical micelle concentration were not toxic to the naphthalene-degrading bacteria and that the presence of surfactant micelles did not inhibit mineralization of naphthalene.

Optimal conditions for polycyclic aromatic hydrocarbon mineralization can be developed by selection of the proper surfactant, bacterial strains, cell and incubation conditions (Willumsen and Karlson , 1998).

A non-sterile biosurfactant preparation () was obtained from a 24-h culture of Bacillus subtilis O9 grown on sucrose and used to study its effect on the biodegradation of hydrocarbon wastes by an indigenous microbial community at the Erlenmeyer-flask scale. Higher concentration gave higher cell concentrations. Biodegradation of aliphatic hydrocarbons increased from 20.9 to 35.5% and in the case of aromatic hydrocarbons from nil to 41%, compared to the culture without biosurfactant. Rapid production of surfactin crude preparation could make it practical for bioremediation of ship bilge wastes (Morán et al., 2000).

48 Literature Review

1.6.2. Enzymes that contribute in PAHs degradation:- 1.6.2.1. Bacterial enzymes:

Since bacteria initiate PAH degradation by the action of intracellular dioxygenases, the PAHs must be taken up by the cells before degradation can take place. Bacteria most often oxidize PAHs to cis- dihydrodiols by incorporation of both atoms of an oxygen molecule. The cis-dihydrodiols are further oxidized, first to the aromatic dihydroxy compounds (catechols) and then channeled through the ortho- or meta cleavage pathways (Smith, 1990).

Among the many different enzymes that are involved in PAH degradation, the initial dioxygenases that enable aerobic bacteria to attack the aromatic ring structures are key enzymes that serve as useful markers for PAH degradation activity. These enzymes are multimeric and are comprised of three components including a reductase, a ferredoxin, and an -sulfur protein (ISPnap) (Simon et al., 1993).

The genes that encode the enzymes involved in the different metabolic steps of aerobic bacterial PAH-degradation pathways have been described in a wide range of Gram negative (GN) bacterial and some Gram positive (GP) bacterial strains (Habe and Omori, 2003). In these conditions, the initial step of the PAH metabolism commonly occurs via the incorporation of molecular oxygen into the aromatic nucleus by a multicomponent aromatic ring-hydroxylating-dioxygenase (RHD) enzyme system forming cis-dihydrodiol (Kauppi et al., 1998).

The naphthalene dioxygenase gene is of particular interest as an indicator for PAH degradation because the enzyme encoded by this gene not only degrades naphthalene, but also mediates degradation of phenanthrene, anthracene, dibenzothiophene, and methylated naphthalenes (Ahn et al., 1999). The nahAc gene also is highly conserved

49 Literature Review

among different Gram-negative bacteria, and this gene in PCR with degenerate primers can detect not only naphthalene-degrading Pseudomonas species but also strains of Mycobacterium, Gordona, Rhodococcus, Sphingomonas that degrade naphthalene and higher molecular weight PAH (Hamann et al., 1999).

Phenanthrene degradation by Pseudomonas mendocina , high level accumulation of the intermediate metabolite 1-hydroxy-2-naphthoic (1H2N) up to 94% of its theoretical was observed. Dynamic profiles of the activities of two key enzymes, i.e. polycyclic aromatic hydrocarbon (PAH) dioxygenase (PDO) and catechol-2,3-oxygenase (C23O), during the biodegradation were revealed (Tian et al., 2002).

PAH dioxygenase (PDO) and catechol 2,3-oxyenase (C23O) are identified as two key PAH-degrading-related enzymes (Meyer et al., 1999). However, to the best of our knowledge, there is no information regarding the simultaneous change of these key enzyme activities in a biodegradation process (Grifoll et al., 1995).

All strains under study with functioning genes of the meta-pathway were shown to possess the activities of the enzymes both of ortho- and meta pathway of catechol oxidation: catechol-1,2-dioxygenase and catechol-2,3- dioxygenase (Filonov et al., 2000).

A dioxygenase gene system, nidA, involved in the initial steps of PAH degradation, commonly was found in known PAH-degrading Mycobacterium strains (Brezna et al., 2003).

Catabolic genes including upper-pathway dioxygenase genes (nahAc and phnAc) and down-pathway catechol dioxygenase genes (C12O and C23O) (Wang et al., 2007).

50 Literature Review

The catalytic action of these enzymes generates more polar and water-soluble metabolites, such as quinones, which are more susceptible to further degradation by indigenous bacteria present in soils and sediments (Meulenberg et al., 1997).

The focus was to analyse the genetic potential and the naphthalene dioxygenase (NDO) expression of the bacterial communities involved in the degradation of naphthalene, as chemical model for the degradation of PAH (Di Gennaro et al., 2009).

Some of these variations are undoubtedly due to differences in the oxidation reduction potentials of the different isoforms (Millis et al., 1989), while some may be caused by slight structural variations at the enzyme active site (Sinclair et al., 1995).

1.6.2.2. Fungal enzymes:

In order for bioremediation to be effective, organisms with PAH degrading enzymes, such as ligninolytic enzyme producers that in turn can convert the substrate(s) into non- or less toxic products, are required (Wu et al., 2008).

The use of ligninolytic fungi. The white rot fungi possess an extracellular degradation system which is capable of breaking down lignin (Kirk and Farrell, 1987), an amorphous and complex biopolymer with an aromatic structure similar to the aromatic molecular structure of some environmental pollutants such as PAHs, pesticides, polychlorinated (PCBs), synthetic dyes, etc. This structural resemblance makes possible the use of white-rot fungi to treat sites contaminated with these recalcitrant compounds (Pointing, 2001).

Several enzymatic mechanisms are thought to be involved: (a) lignin peroxidase (LiP), and possibly also manganese- dependentperoxidase(MnP) , directly catalyze one-electron oxidation of

51 Literature Review

PAHs having the ionization potential (IP) values of < =7.55 eV to produce PAH

quinones (Field et al., 1996) that can be further metabolized via ring-fission (Hammel et al., 1991); (b) catalyzes one-electron oxidation of anthracene and benzo[a]pyrene (both having IP < =7.45 eV), (Collins et al., 1996); some PAH compounds up to six rings were shown to be degradable via MnP-dependent peroxidation reactions both in vitro and in vivo (Bogan and Lamar 1995); (c) intracellular cytochrome P450 monooxygenase activity followed by epoxide hydrolase-catalyzed hydration resulting in hydroxylation of 3-, 4-, and 5-ringed PAHs are believed to initially metabolize PAH molecules including phenanthrene having an IP of 8.03 eV (Bezalel et al., 1997).

Two purified laccase isozymes from T. versicolor were found to have similar oxidative activities towards anthracene and benzo[a]pyrene. was identified as the major end product of anthracene oxidation (Collins et al., 1996).

PAHs’ degradation rates of some white-rot fungi appear not to be correlated with the ligninolytic activities, suggesting that several other enzymetic mechanisms could be used by white-rot fungi to degrade PAHs (Mori et al., 2003).

In contrast, the extracellular ligninolytic enzyme system of the white-rot fungi, consisting of peroxidases and laccases, has been directly linked to biodegradation of PAHs (Wang et al., 2009).

Bjerkandera sp. produced MnP (Field et al., 1995), and P. chrysosporium produced MnP and LiP (Bogan et al., 1996a) as shown on the level of gene transcription. Ectomycorrhizal fungi released free extractable phenoloxidases, PO, MnP, mono- and dioxygenases, as well as oxidases that generate , the cosubstrate of peroxidases (Gramss 1997). Many of the PAH oxidizing enzymes were also released

52 Literature Review by mycelia of those wood- and straw-degrading and terricolous basidiomycetes (Gramss, 1979).

The Chilean white-rot fungus Anthracophyllum discolor produces high levels of (MnP) in the presence of wheat grains as lignocellulosic support (Rubilar, 2007), and to a lesser extent laccase (L) and lignin peroxidase (LiP), and is efficient in the degradation of organic pollutants such as chlorophenols and dyes (Tortella et al., 2008).

Laccase of Trametes versicolor was generally able to oxidize anthracene in vitro (Johannes et al., 1996).

Enzymatic assays showed that laccase and manganese independent peroxidase activity could have played a role in the degradation process (Anastasi et al., 2009).

These extracellular ligninolytic enzymes, laccases, manganese dependent peroxidases (MnP), and lignin peroxidases (LiP), with very low substrate specificity to break down the irregular structure of lignin. These enzymes play a very important role in the biodegradation of various chemical pollutants (Wang et al., 2009).

As a result of the low specificity of ligninolytic exoenzymes; laccase, manganese peroxidase, and lignin peroxidase, various strains of white-rot fungi are able to degrade complex aromatic compounds such as , polychlorinated compounds (PCBs), pentachlorophenol, and PAHs (Kamei et al., 2006).

Laccase and manganese peroxidase activities, but not lignin peroxidase activity, were detected during the biodegradation of fluorene. Two of the metabolites from fluorene degradation by the fungus were identified via reversed-phase HPLC as 9-fluorenol and 9-, the less toxic intermediates of fluorene. However, 9-fluorenol is not an end product for the

53 Literature Review degradation. These results suggest that fluorene was degraded by Agrocybe sp (Chupungars et al., 2009).

Extracellular enzymes secreted by A. fumigatus could metabolize anthracene effectively, in which the lignin peroxidase may be the most important constituent (Ye et al., 2011).

1.7. Pathway for PAHs degradation:

Salicylic acid, an intermediate compound formed in the pathway of microbial degradation of naphthalene (Reshetilov et al., 1997) as indicated in Fig. (1).

In case of Naph. biodegradation, the natural isolates with functioning genes of the ortho-pathway and silent genes of meta cleavage of catechol oxidation were less than for isogenous strains with the functioning genes of the meta-pathway. All strains under study with functional genes of the meta-pathway were shown to possess the activities of the enzymes both of ortho- and meta pathway of catechol oxidation: catechol-1,2-dioxygenease and catechol 2,3 – dioxygenase (Filonov et al., 1999 and 2000).

Streptomyces griseus catalyzed the biotransformation of naphthalene to 4-hydroxy-1-tetralone in a good yield. The intermediates formed also were 2-Methyl-1-4- Naphoquinone and 2-Methyl-4-hydroxy- 1-tetralone (Gopishetty et al., 2007) as indicated in Fig. (2).

Existence of nahAc and C23O was confirmed in the system and the copies of the two genes in the aerobic tank were 2 or 3 orders higher than those in the influent water sample. The different behavior of C23O demonstrated that mineralization of PAHs might mainly occur in the aerobic unit (Wang et al., 2007).

54 Literature Review

Figure (1): Proposed pathway for the degradation of naphthalene by Pseudomonas putida (Reshetilov et al., 1997)

Figure (2): Proposed pathway for the degradation of naphthalene by Streptomyces griseus (Gopishethy et al., 2007)

55 Literature Review

The metabolic pathway of phenanthrene by bacteria especially Pseudomonas was well studied and most bacteria utilized dioxygenase enzymatic system for initial attack and transformed to dihydroxy phenathrene (Sutherland et al., 1990).

As a result of oxidation at the C-9 and C-10 positions, a ring- fission product 2,2 – diphenic acid be produced as an intermediate (Hammel et al., 1992).

Phenanthrene metabolism by Pseudomonas sp. in soil is known to produce pyruvate as an intermediate product (Juhasz et al., 1997).

It was reported that phenanthrene degradation by Nocardioides might follow either or salicylic pathways. Phenanthrene → 1- Hydroxy-2-Naphthoic acid → 1-Naphthol (salicylate route) → → Catechol → CO2 + H2O (Iwabuchi and Harayama, 1997).

Genes that responsible for degradation of organic pollutants may be in plasmid rather than the chromosome (Anokhina et al., 2004).

It may be mentioned that 2-Naphthol, a decarboxylated product of 2HINA was detected as a minor metabolite in the degradation of phenantherene by Staphylococcus sp. strain PN/Y. Moreover, it has been observed that 2-Naphthol was toxic intermediate and the minimum growth inhibitory concentration was found to be 45 mg /l (Mallick et al., 2007).

Toxicity of 2-naphthol has also been reported earlier in Burkholderia and Pseudomonas spp. (Balashova et al., 1999).

Shingomonas sp. GY2B could efficiently degrade phenanthrene at 100 mg /1 as the sole carbon source and that it could also degrade other

56 Literature Review aromatic compounds such as phenanthrene and 1-Hydroxy-2-Naphthoic acid (Tao et al., 2007) as indicated in Fig. (3).

Staphylococcus sp. PN/Y degraded phenanthrene by a novel pathway involving 2-hydroxy-1-naphthoic acid (2H1NA), which was further metabolized by unique meta-cleavage dioxygenase, ultimately leading to TCA cycle intermediates (Mallick and Dutta, 2008).

Identification of phenanthrene-degrading gene in PAH-degrading bacteria generally exhibit the same degradation pathway. Phenanthrene is initially transformed to cis-dihydrodiol by PAH dioxygenase (a multicomponent of dioxygenase enzyme system); dihydrodiol converts dihydrodiol to catechol; and, then, catechol is degraded into or acids by catechol 2,3-dioxgenase (Haritash and Kaushik, 2009) as indicated in Fig. (4) and (5).

Hammel et al. (1992) reported in 1992 that Phanerochaete chrysosporium metabolizes phenanthrene 9,10-quinone and 2,9-diphenic acid to form diphenic acid and eventually CO2.

57 Literature Review

Figure (3): Proposed pathway for the degradation of phenanthrene by sphingomonas sp. (Tao et al., 2007).

Figure (4): Postulated metabolic pathway of PAH-degradation in aerobic bacteria. Enzymes involved in the degradation of PAHs are oxygenase and dehydrogenase (Haritash and Kaushik, 2009). The initial PAH dioxyganase and catechol 2,3-dioxygenase are encoded by nahA and nahH, respectively.

58 Literature Review

Figure (5): Proposed phenanthrene degradation pathways by the managrove enriched bacterial consortium (Luan et al., 2006).

59 Literature Review

The formation of phenanthrene trans-9R, 10R-dihydrodiol, in which only one of oxygen originated from molecular oxygen, indicates that P. ostreatus initially oxidizes phenanthrene stereoselectively, via a cytochrome P-450 monooxygenase and an epoxide hydrolase rather than a dioxygenase, to form the dihydrodiol as indicated in Fig. (6) (Bezalel et al., 1996).

The intermediate reported by Phen. degradation by bacteria follow either phathalate or salicylate pathway. Formation of 1-Hydroxy-2- naphthoic acid, 1-Naphthol, salicylic acid catechol to give finally CO2 and

H2O (Tao et al., 2007).

Phenanthrene Phenanthrene Phenanthrene trans 9,10- 9,10-dihydroxy 9,10-phenanthrene 2,2'-Diphenic acid 9,10-oxide dihydrodiol phenanthrene quinone Figure (6): Proposed pathway for the degradation of phenanthrene by (Bezalel et al., 1996).

The degradation of anthracene outcomes into its total decomposition to the dead-end product: anthraquinone. The degradation mechanism, probably arising via one-electron oxidative pathway, has a large complexity with the generation of intermediate compounds such as anthrol and anthrone (Haemmerli, 1988).Anthracene could be oxidized by a nucleophilic attack at either position 9 or 10, due to the high charge at these positions, resulting in the formation of a C centered radical which would undergo further spontaneous nonenzymatic rearrangements to form 9,10-anthraquinone (Collins et al., 1996).

60 Literature Review

Degradation of anthracene by Pseudomonas sp. was reported by hydroxylate aromatic ring transformation to cis-1,2-dihydroanthracene- 1,2-diol which was further converted to anthracene-1,2-diol (Cernigia, 1984). This upon cleavage at meta position yielded 4-(2-hydroxynaph-3- yl)-2-oxobut-3-enoate which rearranged to form 6,7-benzocoumarin or be converted to 3-hydroxy-2-naphthoate, from which degradation proceeded through 2,3-dihydroxynaphthalene to salicylate (Akhtar et al., 1975). At present, the only known productive pathway for bacterial degradation of anthracene, proceeds through 3-hydroxy-2-naphthoic acid, 2,3- dihydroxynaphthalene and further through a pathway similar to the naphthalene degradation pathway (Tongpim and Pickard, 1999). Degradation from anthracene to 3-hydroxy-2-naphthoic acid proceeds through dioxygenation and dehydration, by which 1,2- dihydroxyanthracene was formed which is further cleaved by meta-ring cleavage to 2-hydroxy-3-naphthaldehyde and then to 2-hydroxy-3- naphthoic acid leading to side product 6,7-benzocoumarin (Moody et al., 2001).

Phthalic acid was reported as an oxidation product of anthracene from Bjerkandera sp. BOS55. the degradation products for fluoranthene were 4-hydroxy-9-fluorenone and 9-fluorenone and for pyrene, it was 4,5- dihydropyrene. In addition trans-4,5-dihydrodiolpyrene has been reported as a degradation product of pyrene by several white-rot fungi (Sack et al., 1997; Cajthmal et al., 2002 and Eibes et al., 2006).

Anthracene transformation occurred throughout the 25-day course of the experiment and, therefore, likely involves mechanisms distinct from those involved in oxidation of non-LiP substrate polycyclic aromatic hydrocarbons (Bogan et al., 1996b).

61 Literature Review

In this work, we showed that C. elegans, A. terreus, A. fusca, and A. cylindrospora produced 1-4 dihydroxyanthraquinone as major product. However the cytotoxicity observed with the dihydroxyanthraquinones was considerably reduced compared to that exhibited by anthracene (AC). In Tetrahymena pyriformis, the 1-4 dihydroxyanthraquinone exhibited an higher inhibitory effect than anthracene (AC) on the population growth rate (Guiraud et al., 2008).

An anthracene-degrading strain, identified as Aspergillus fumigatus, showed a favorable ability in degradation of anthracene. The degradation efficiency could be maintained at about 60% after 5 d. with initial pH of the medium kept between 5 and 7.5, and the optimal temperature of 30ºC as indicated in Fig. (7) (Ye et al., 2011).

Figure (7): Proposed pathway for the degradation of anthracene by Aspergillus fumigatus (Ye et al., 2011).

The initial dioxygenase (PDO) is responsible for the first step in the aerobic degradation of polyaromatic compounds, catalyzing the hydroxylation of the substrate to the corresponding cis-dihydrodiol (Resnick et al., 1996). The catalytic meta-cleavage of catechol by C23O

62 Literature Review seems to be the most common pathway in the subsequent steps of PAH degradation (lower pathway) (Meyer et al., 1999).

Aerobic metabolism is by far more efficient for oxidation of organic molecules than anaerobic metabolism due to the abundance of O2 serving both as an electon acceptor and a source of oxygen (Quantin et al., 2005).

Kim et al. (2005) found that detected lactone compound and 4- hydroxy phenanthrene in the pyrene degradation by Mycobacterium vanbaalenii PYR 1. Nevertheless, these two metabolites have been first described in the degradation of PAHs by bacterial consortium consisting of Rhodococcus sp., Acinetobacter sp. and Pseudomonas sp.

It is possible that a bacterial consortium consisted of different strains could utilize both dioxygenase and mono-oxygenase systems to transform pyrene to cis-pyrene or trans-pyrene dihydrodiol, and further to dihydroxy pyrene and monohydroxy pyrene, respectively. However, only cis-4,5-dihydroxypyrene was detected in the present study, suggesting that the mangrove enriched bacterial consortium utilized dioxygenase system to transform pyrene. Dihydroxy pyrene was further degraded to lactone and then further to 4-hydroxyphenanthrene as indicated in Fig. (8) (Luan et al., 2006).

The results of pyrene degradation indicated that a number of acidic intermediate products such as succinic acid, , and be produced in the degradation process of PAH. The pH decline resulted from the accumulation of those acid intermediate products. And this may be the reason that the change in pH corresponded to the change in PAH concentration (Lin and Cai, 2008).

63 Literature Review

Figure (8): Proposed pyrene degradation pathways by the mangrove enriched bacterial consortium (Luan et al., 2006).

Pyrene degradation by Mycobacterium flavescens was indicated in Fig. (9) and by Mycobacterium sp. strain PYR-1 was indicated in Fig. (10). However, pyrene degradation by the fungal Aspergillus niger SK9317 was indicated in Fig. (11).

64 Literature Review

Figure (9): Proposed pathway for the degradation of pyrene by Mycobacterium flavescens (Dean-Ross and Cerniglia, 1996).

Figure (10): Proposed pathway for the degradation of pyrene by Mycobacterium sp. strain PYR-1 (Cerniglia, 1992)

65 Literature Review

Figure (11): Proposed pathway for the degradation of pyrene by Aspergillus niger SK 93/7 (Wunder et al., 1994). Benz(a) anthracene-7, 12-dione was degraded to 1,2- naphthalenedicarboxylic acid and phthalic acid that was followed with production of 2-hydroxymethyl or monomethyl and dimethylesters of phthalic acid. Another degradation product of BaAQ was identified as 1-tetralone. Its transformation via 1,4-naphthalenedione, 1,4-naphthalenediol and 1,2,3,4-tetrahydro-1-hydroxynaphthalene resulted again in phthalic acid. None of the intermediates were identified as dead- end metabolites. Metabolites produced by ring cleavage of benz[a] anthracene using the ligninolytic fungus are firstly presented in this work (Cajthmal et al., 2006) as indicated in Fig. (12).

At least two dihydrodiols, 5,6-BAA-dihydrodiol and 10,11-BAA- dihydrodiol, were confirmed by high-resolution mass spectral and fluorescence analyses as products of the biodegradation of BAA by Mycobacterium sp. strain RJGII-135 as indicated in Fig. (13) (Schneider et al., 1996).

66 Literature Review

1: BaAQ; 2: 1-tetralone; 3: 1,2,3,4-tetrahydro-1-hydroxynaphthalene; 4: 4-hydroxy-1-tetralone, 5: phthalic acid; 6: Phthalic acid monomethyl 7: 2-hydroxymethyl benzoic acid; 8: dimethylesters; 9: 1,4-naphthalenedione; 10: 1,4-dihydroxynaphthalene; 11: 1,2-naphthalenedicarboxylic acid.

Figure (12): Proposed pathway for the degradation of benzo [a] anthracene by the ligninolytic fungus Irpex lacteus (Cajthmal et al., 2006).

Figure (13): Proposed pathways for the degradation of [B-a-Anth.] by Mycobacterium sp. strain RJGII-135 based on isolated metabolites (Schneider et al., 1996).

67 Materials and Methods

MATERIALS AND METHODS

68 Materials and Methods

2. MATERIALS AND METHODS

2.1. Materials: 2.1.1. Sampling sites: Soil and sludge samples were collected from deposits of petroleum field which are either chronic or recent from the Cairo oil Refining company, Al-Qalyubiyah, Egypt as indicated in Fig. (14).

Mostorod

Figure (14): Sampling site map.

2.1.2. Sampling: Soil samples and sludge of waste water were collected from 5 to 30 cm below the surface with sterilized soil cores and the top 5 cm of the samples were discarded. The soil cores were placed in sterile bags, shipped on ice and stored at 4oC to be used within 4 hours. Water samples were collected in 250 ml screw capped sterile glass bottles and transported on ice.

69 Materials and Methods

Table (1): Sampling sites represented different sources of indigenous microbial communities

Sample Type of soil Depth Distance from the origin History of PH Site in (No.) deposit exposure Egypt

1 Soil contaminated with oil Zero m * Chronic soil 4.99 surface

2 Soil contaminated with oil Zero m * Recent soil 4.74

surface Mostorod -

3 Soil contaminated with oil Zero m * Chronic soil 5.40 30cm ˙

4 From drainage of waste 200 m * Chronic sludge 4.90 Qalyubiyah

petroleum - 30 cm ˙ Al 5 Soil contaminated with oil 300 m * Recent soil 5.40 30 cm ˙

6 Soil contaminated with oil 200 m * Chronic soil 5.11 surface Cairo Oil Refining Company Oil Refining Cairo 7 Agriculture soil Surface 400 m * Chronic soil 5.49 * m = meter ˙cm = centimeter

70 Materials and Methods

2.1.3. Media:

2.1.3.1. Basal salt medium (BSM): (Ogawa and Miyashita, 1995).

This medium consist of the following ingredients (g/L).

(NH4)2 SO4 1.1

K2 HPO4 2.2

K H2 PO4 0.9

Mg.SO4. 7 H2O 0.1

Mn. SO4 . 6 H2O 0.025

Fe SO4. 7 H2O 0.005 L-Ascorbic acid 0.005 Deionized water 1000.0 ml

For use, the following supplements were added to 1 liter of the cooled basal medium.

 1 ml of trace element.

 0.1 ml of solution

Trace element: mg/L

H3 BO3 0.3

Co SO4 0.4

Zn SO4. 7 H2O 0.1

MnCl2 . 4 H2O 0.03

NaMo O4 . 2 H2O 0.03

Ni SO4 . 6 H2O 0.02

Cu SO4 . 5 H2O 0.01 HCl 50 ml Deionized water 950.0 ml

71 Materials and Methods

Vitamin Solution: mg/L Biotine 2.0 Folic acid 2.0 Pyridoxal hydrochloride 10.0 Riboflavine 5.0 Thiamine 5.0 Nicotonic acid 5.0 Ca-panthothenate 5.0 Cyanocobalamine 5.0 P-aminobenzoic acid 5.0 Deionized water 1000.0 ml

2.1.3.2. Luria-Bertani (L.B) broth medium (Martin et al., 1981)

This medium consists of the following ingredients (g/L): Tryptone 10.0 extract 5.0 NaCl 5.0 Distilled water 1000.0 ml

2.1.4. pH determination: (Fulthorpe et al., 1996)

For each sample, the pH determination has done. A slurry was made by vortexing 1 gram of soil in 5 ml of deionized water for 1 minute.

2.1.5. Protein determination: (Lowry et al., 1951)

To determine the amount of soluble protein in any culture of the polycyclic aromatic hydrocarbon degrading bacteria, the following solutions must be prepared. Sol. (A) Cupper sulphate 1.0%

72 Materials and Methods

Sol. (B) tartrate 2.0% Sol. (C) Sodium 2.0% + 0.4%

Five ml of the reaction solution was added to 1 ml of the diluted sample of the culture filtrate. Distilled water was used as a blank. Then the mixture was allowed to stand at room temperature for 10 minutes. After that 0.5 ml of Folin was added. The reaction tubes were incubated at room temperature for 20 minutes. The absorbance was determined at 720 nm. To determine the concentration of the protein in samples, a standard curve of Bovine serum albumin (BSA) was determined.

2.1.6. Chemicals:

Pyrene and fluoranthene from Sigma-Aldrich, USA, Anthracene, from Alfa Aesar, Jermany.

Folin reagent product of Sigma (Aldrich, USA).

Benzo (a) anthracene and phenanthrene 90%, from AcRòs organics, New Jersey, USA. Naphthalene, from ADWIC, ElNasr Pharmaceutical chemicals co., Egypt.

Acenaphthene, from Schroelzbereich Siedebereich.

Chloroform, and were HPLC grade, obtained from BDH, England.

Bovine serum albumen (BSA) obtained from Sigma, (St. Louis), USA.

2.1.7. Bacterial strain:

Enterobacter cloacae MAM-4 isolated from waste water contaminated with heavy metals, Cairo, Egypt. This strain was kindly provided by Dr. Mervat Abo-State

73 Materials and Methods

2.1.8. Source of gamma radiation:

The Indian chamber of -60 located at the National Center for Radiation Research and Technology (NCRRT), Nasr City, Cairo, Egypt was used for the irradiation treatment. The dose rate was 1 KGy/15 minutes at the time of experiment at room temperature.

2.1.9. High performance liquid chromatography (HPLC):

The quantitative determination of various polyclic aromatic hydrocarbons (PAHs) compounds was performed using High performance liquid chromatography (HPLC) in Egyptian Petroleum Research institute, Cairo, Egypt.

The various PAHs were quantified by (HPLC pump No. 2360, gradient programmer No. 2360 and detector No. UA-5 with a 280 nm fitter (Isco, Inc.); Integrator No. SP4600 [Spectra-Physics] and HPLC autosampler No. 738 [Alcott chromatography] with the 150 mm reversed phase column hypersil ODS-C18, 5 µm [Altech; No. 9876]. The mobile phase consisted of methanol water and 0.5% acetic. The methanol/ water ratio varied from 70:30 to 5:95 (Utkin et al., 1995).

2.1.10. / (GC-MS):

The qualitative and quantitative determination of various polycyclic aromatic hydrocarbon (PAH) compounds was performed using Gas Chromatographic/Mass Spectrometry (GC/MS) in Central Laboratory Holding Company for Water and Waste-Water; Cairo, Egypt.

The GC is a 3800 Varian USA, EI-ITS 1200 L Varian USA (Electron Impact Source, Quadrupole MS and EMD Detector). The capillary column was a VF-5-MS Capillary (30 m x 0.25 mm). Helium 5.0 was used as carrier gas for the system (75 Psi 1 ml min-1). The chromatographic temperature programme for GC/MS was: Start (t = 0) at 60oC followed by a 10oC min-1 increase to 160oC and 4-250oC maintaining this final temperature

74 Materials and Methods for 10 min. Temperature of the injector was set to 250oC transfer line: 270oC. The injection volume was 1 µl in the splitless mode.

A measured volume of sample, 1 Liter, is serially extracted with methyle dichloride at pH greater than 11 and again at pH less than 2 using seperatory funnel or continous extractor. The dichloride extract is dried, concentrated to a volume 1 ml, and analyzed by GC/MS.

Qualitative identification of the compounds (standard and intermediates) in the extract is performed using the retention time and the relative abundance of three characteristic masses (m/z).

Quantitative analysis is performed using internal standard technique with a single characteristic (m/z) (Hung and Thiemann, 2002).

2.2. Methods:

2.2.1. Cultivation of samples in basal salt medium (BSM):

Soil samples and sludge (25 grams) or (25 ml) of water samples were added to (100 ml) of BSM and incubated overnight in shaking incubator at 30oC with 150 rpm for adaptation of the microbial communities (Indigenous mixed bacteria). Then the solid particles were allowed to sediment, to be used for inoculation of BSM later on (Juhasz and Naidu, 2000).

2.2.2. Growth of different microbial communities on different polycyclic aromatic hydrocarbon compounds:

From the preadapted microbial communities, 10.0 ml was used to inoculate 100.0 ml of BSM. The BSM was amended by 50 mg/L naphthalene (Naph.) 250 mg/L phenanthrene (Phen.); 50 mg/L anthracene (Anth.), 100 mg/L acenaphthene (Ace.), 10 mg/L fluoranthene (Flu.), 100 µg/L pyrene (Pyr.) and 100 µg/L benzo (a) anthracene (B-a-anth.). Three replicates were used for each treatment.

Growth was determined by measuring optical density (O.D) at 600 nm periodically at zero time (initial), 7, 15, 21 and 28 days. Extracellular

75 Materials and Methods protein was determined at 720 nm periodically at zero time (initial), 7, 15, 21 and 28 days using spectrophotometer (LW-V-200 RS UV/VIS, ). Also, the bacterial count (CFU/ml) by spreading the serially appropriate diluted cultures on L.B agar medium at the beginning (Io) and after 28 days incubation (I) have been determined. The inoculated plates was incubated at 37oC for 48 hours.

2.2.3. Determination of total bacterial count and hydrocarbone degrading bacterial (HDB) count:

The pre-adapted microbial communities of the different sources were serially diluted and the appropriate three successive dilutions were plated on L.B agar plates and BSM agar plates amended with 1.0% crude petroleum oil. The inoculated plates were incubated at 30oC for 48 hours for L.B plates and for 7 days for the hydrocarbon degrading bacterial (HDB) plates. The bacterial count was determined.

2.2.4. Isolation of different bacterial strains capable of growing on hydrocarbons:

The well grown bacterial colonies on BSM amended with crude petroleum oil were picked up as separated single colonies. These colonies were called HDB. They stored on slants of L.B medium for further investigation at 4oC.

2.2.5. Screening for the most promising indigenous hydrocarbon degrading bacterial (HDB) isolates on PAHs:

The well grown HDB isolates which are fourty four strains, were streaked on BSM agar plates amended with 500 mg/L Naph., 100 mg/L Anth., 500 mg/L Phen.; 250mg/L Ace.; 20 mg/L Flu.; 150 µg/L B-a-Anth. and 150 µg/L Pyr. Three replicates were used for each strain on each compound. The plates were incubated at 30oC. The growth was shaked every day for 15 days.

76 Materials and Methods

22.6. Characterization of the most promising polycylic aromatic hydrocarbon degrading bacterial (PAHDB) isolates:

The six most promising PAHD isolates (MAM-26, MAM-29, MAM- 43, MAM-62, MAM-68 and MAM-78) and Enterobacter cloacae MAM-4, that have the ability to grow on the different concentrations of different PAH compounds were characterized and investigated for Gram stain with light microscope (leica, LEITZ; LABOR LUXS, Germany).

2.2.7. Determination of the best isolate has the ability to degrade different polycyclic aromatic hydrocarbon compounds:

The six most promising PAHDB and the standard isolate E. cloacae MAM-4 were grown on L.B broth media for 48 hours in shaking incubator (150 rpm) at 30oC. The well grown cultures were centrifuged at 8000 rpm for 10 minutes. The pellets were washed twice with sterile BSM. The washed pellets were suspended in BSM supplemented with PAH compounds and incubated in shaking incubator (150 rpm) at 30oC for 3 days for adaptation.

Fifteen ml of each of the pre-adapted seven selected isolated bacterial strains was used to inoculate 150 ml of BSM. The BSM was amended by five different concentrations of the five selected PAH compounds [(Naph. 500, 750, 1000, 1500 and 2000) mg/L, (Phen. 250, 500, 750, 1000, 1500) mg/L, (Anth. 40, 50, 75, 100, 150, 200, 300, 400) mg/L, (Pyr. (100, 200, 300, 400, 500 µg/L) and (B-a-anth., 100, 200, 300, 400 and 500) µg/L]. Three replicates were used for each strain inoculated in each BSM containing compound for each concentration.

Growth was determined by measuring optical density (O.D) at 600 nm periodically at zero time (initial), 1, 2, 3, 4, 5, 6, 7, 14 and 21 days using spectrophotometer LW-V-200 RS UV/VIS, Germany).

Also protein was determined at 720 nm periodically at zero time (initial), 1, 2, 3, 4, 5, 6, 7, 14 and 21 days using spectrophotometer (LW-V- 200 RS UV/VIS, Germany).

77 Materials and Methods

Quantitative analysis by HPLC were determined at the end of incubation period (21 days).

Also bacterial count was determined at zero time (initial) and 21 days.

2.2.8. Identification of the most promising isolated strain (wild strain):

2.2.8.1. Phenotypic characterization of PAH degrading bacterial strains:

Colony morphology of the isolated most potent polycyclic aromatic hydroxation degrading strain (MAM-29 and MAM-62 one as gram -ve and the other as gram +ve) was assessed by monitoring their growth on L.B agar plates. Cellular morphology was examined by light microscope (Leica, LEITZ, LABOR LUXS, Germany).

2.2.8.2. DNA extraction:

Genomic DNA was extracted from pure bacterial culture; 24 hr grown in Luria-Bertani (L.B) medium at 30oC, centrifuged for 2 min. Bacterial lysis was performed according to the manufacturer's instructions using The GeneJETTM genomic DNA purification kit (Fermentas life sciences, EU). The obtained purified DNA was re-suspended in 100 l of TE buffer (Sambrook and Russel, 2001).

2.2.8.3. PCR amplification of bacterial 16S-rRNA:

Oligonucleotide primers were used to amplify 16S-rRNA. The universal primers: PA forward: AGAGTT TGATCCTGGCTCAG and PH reverse: AAGGAGGTG ATCCAGCCGCA (synthesized in Korea) were used to amplify the 16S-rRNA. 16S-rRNA was amplified from the obtained DNA in a reaction mixture of PCR conditions were as follows: 10xTaq buffer, 1.25 U AmpliTaq Gold DNA Polymerase (Fermentas,

EU), 2mM dNTP mixture, 25mM MgCl2, 0.7 g DNA, double-distilled

78 Materials and Methods

water mixed in a final volume of 50l. The program for PCR was as follows: the initial denaturation at 95oC for 5 min, followed by 30 cycles of 95oC for 1 min, annealing at 55oC for 1 min, and 72oC for 2 min, and final extension at 72oC for 7 min. (Edwards et al., 1989). Amplification was done using Perkin Elmer GeneAmp PCR system 2400 (Germany). Analysis of the PCR products was performed by electrophoresis on 1% agarose gels using standard conditions according to Sambrook and Russel (2001).

2.2.8.4. Cloning and sequencing:

16S-rRNA PCR product was extracted from gel using gel extraction kit QIAquick Qiagen (Promega, USA). DNA sequencing was conducted using ABI Prism BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, USA) according to manufacturer's instructions. ABI PrismTM 3730/3730XL DNA Sequencer (AME Biosciences, USA).

2.2.8.5. Phylogenetic analysis:

The 16S-rRNA DNA sequence was submitted to the National Center for Biotechnology Information (NCBI) database and the sequence was compared to other available 16S-rRNA sequences using an automatic alignment tool (Blastn). The construction of the phylogenetic tree was generated by PhyML and the visualization of the tree by TreeDyn using the online program www.phylogeny.fr. The bootstrap values were obtained by drawing a tree.

2.2.8.6. Nucleotide sequence accession number:

The 16S-rRNA sequence was deposited in the NCBI Gene Bankit nucleotide sequence database under accession number JN038054 for (MAM-62) and JN038055 for (MAM-29).

79 Materials and Methods

2.2.9. Effect of gamma irradiation on the viability of the selected isolated bacterial strains:

The most promising selected isolated bacterial strain (MAM-62) was grown in L.B broth medium for 24 hours at 37oC in shaking incubator (200 rpm). The well grown bacterial cells were harvested by centrifugation at 8000 rpm for 10 minutes, washed with sterile saline, and resuspended in the sterile saline.

Cells suspended in saline were distributed into 5 ml aliquots in sterile screw capped test tubes, and then exposed to different doses of gamma irradiation (Indian cell – 60Co) with dose rate 1 kGy/15 min., in National Center for Radiation Research and Technology (NCRRT), Nasr City, Cairo, Egypt.

Three replicates were used for each dose. Survival of these bacterial strains were determined (Dose response curve) on L.B agar plates.

2.2.10. Selection of the best mutant has the ability to grow on different polycyclic aromatic hydrocarbon compounds:

Colonies exposed to different doses of gamma irradiation were picked up from the L.B agar plates. The irradiated colonies, which showed any difference in their morphological characters (shape, color, margin, surface, size …, etc) were collected.

The 24 irradiated colonies and the non-irradiated control (parent strain) were grown each in 50 ml L.B broth and incubated at 30oC for 48 hours in shaking (150 rpm) incubator. The grown cultures were centrifuged at 8000 rpm for 10 min. the pellets were washed twice with sterile BSM.

The washed pellets were suspended in BSM and used to inoculate (10% v/v) BSM amended with PAH compounds (1000 mg/L Naph.; 750 mg/L Phen.; 75 mg/L Anth.; 300 ug/L Pyr. and 300 µg/L B-a-Anth.). Three replicates were used for each treatment. Growth was determined by measuring O.D at 600 nm at the initial ,1st ,2nd and after 7 days.

80 Materials and Methods

2.2.11. Comparative study between the wild strain and the mutant strain on their degradability of the different polycyclic aromatic hydrocarbon compounds:

Each of the selected promising wild strain and the mutant strain were grown in L.B broth medium for 24 hours at 37oC in shaking incubator (150 rpm). The well grown bacterial cells were harvested by centrifugation at 8000 rpm for 10 min., washed with BSM, and resuspended in the BSM.

10 ml was used to inoculate 100 ml of BSM. The BSM which was inoculated by 10 ml was used to inoculate 1000 ml of BSM amended by 1000 mg/L naphthalene, 750 mg/L phenanthrene, 75 mg/L for anthracene and 300 µg/L for pyrene and B-a-anthracene. Three replicates were used for each treatment.

Qualitative and quantitative analysis by gas chromatographic/mass spectrometry (GC/MS) were determined after 24 hours of incubation.

81 Results and Discussion

RESULTS AND DISCUSSION

82 Results and Discussion

3. RESULTS AND DISCUSSION

3.1. Growth of different indigenous bacterial communities on different PAHs.

The soil and sluge samples collected from Cairo Oil Refining Company and Agriculture . Soil near this company have been used to isolate their microbial communities (Indigenous mixed bacteria) to investigate their ability to grow and degrade the chosen polycylic aromatic hydrocarbons [naphthalene (Naph.), Phenanthrene (Phen.), anthracene (Anth.), Acenaphthene (Ace.), Fluroanthen (Flu.), Pyrene (Pyr.) and Benzo-a- anthracene (B-a-Anth.] as a sole carbon and energy source.

The seven different indigenous microbial (bacterial) communities as indicated in Table (2) were isolated from recent and chronic soils contaminated with petroleum at different depths and distances. The chronic soil had a 41 years exposure history for deposition of petroleum wastes. While the agriculture soil had in addition to contamination of with petroleum hydrocarbons a history of exposure to pesticides.

Growth of bacterial community (1) was the best growth on 500 mg/L Naph. As indicated in Table (2) and Figure (15). The growth of this community was 5.0 times the initial after 7 days incubation this growth continued its increase to be 10.0 times the initial after 15 days incubation, then began to decrease at 21 and 28 days to be 9.5 and 8.6 times respectively.

The extracellular protein of community (1) increased to be 2.7 times the initial after 7 days incubation. This represent the highest extracellular protein secreted by this community. As, the incubation period of community (1) increased more, the secreted protein decreased as indicated in figure (16).

83 Results and Discussion

Community (2) was the second in growth, as the incubation period increased from 7 days to 15 days, the growth increased from 4.1 to 6.8 times the intitial. However, community (2) was the most extracellular protein producer in all communities. Its increase was 11.0 times of the initial after 7 days incubation period.

The ability to community (1) to growth on 250 mg/L. phenanthrene was 2.8, 2.7, 4.6 and 6.4 times the intitial after 7, 15, 21 and 28 days respectively as indicated in Table (3) and Figure (17). This means that community (1) needs more time to continue its growth increase.

The present study suggested two possible reasons for the different persistence of LMW and HMW PAHs: The PAH degrading bacteria in polluted environment degrade LMW PAHs faster; (2) LMW and HMW PAHs are degraded by different bacterial groups in the environment, and the abundance and activity of the two bacterial groups affect the biodegradation (Zhou et al., 2008)

Muller et al. (1998) Isolated microorganisms able to convert naphthalene, anthracene and phenanthrene under thermophilic conditions. Studies indicate that metabolites differ significantly from those formed under mesophilic conditions.

Under anaerobic conditions, rates of degradation of PAHs may be negligible (Baur and Capone, 1985).

The availability of oxygen exerts a strong influence on degradation rates, with degradation rates being zero or very slow without oxygen (Mille et al., 1988).

In addition, organisms that Proliferate at elevated temperatures (thermophiles) might have inherently higher PAH degradation rates, since substrate utilization rates of thermophiles have been observed to be 3-10

84 Results and Discussion times higher than those observed with analogous mesophilic bacteria (Lapara and Allenman, 1999).

Our results suggest that optimum conditions for PAH mineralization can be achieved by selection of the proper surfactant, bacterial strain and incubation conditions (Willumsen and Karlson., 1998).

Microbial mineralization of polycyclic aromatic hydrocarbons (PAHs) in soil has been shown to decrease as PAH residence time increases (Hatzinger and Alexander, 1995). Interactions between the PAHs and soil organic matter (SOM) are believed to be responsible for the decline in degradation over time. These interactions include partitioning (Pignatello and Xing, 1996), adsorption and absorption (Weber and Huang, 1996), chemisorptions (Maruya et al., 1996), diffusion, dissolution (Ehlers and Luthy, 2003), and covalent binding (Bollag, 1992), which result in an aged or defined as the movement of chemical into soil micropores or into the soil organic matrix where humin pore sizes range from 2 to 360 nm (Malekani et al., 1997) and the transformation and/or incorporation of pollutants into stable soil solid phases (Ehlers and Luthy, 2003). This process limits the release of PAHs into the bulk liquid phase, making them inaccessible to microorganisms, thus decreasing biodegradation rates (Hatzinger and Alexander, 1995; Willumsen and Karlson, 1997).

When present in mixtures, PAHs have the capacity to influence the rate and extent of biodegradation of other components of the mixture. In some cases, these interactions may be positive, resulting in an increase in biodegradation (Dean-Ross et al., 2002).

Wen et al. (2009) indicated that the enzyme activities and biodegradation rates are influenced by pH, tempature, substrate concentration, the presence of substrates/ mediators.

The degradation efficiency could be maintained at about 60% after 5 d with initial pH of the medium kept between 5 and 7.5, and the optimal

85 Results and Discussion temperature of 30c. The activity of some strains was not affected significantly by high salinity (Ye et al., 2011).

Some bacteria produce biosurfactants which increase the solubility of PAH compounds, while other microorganisms enhance cell surface hydrophobicity or form biofilms to facilitate growth on hydrophobic compounds, and ligninolytic fungi excrete enzymes (Doyle et al., 2008).

In this context, Ijah and Antai (2003) reported that Bacillus sp. were the predominant microorganisms in highly polluted soil samples.

During active contaminant biodegradation, microbes that use the contaminants as carbon and energy sources increase in number (Ringelberg et al., 2001). This increase in the amount of contaminant-degrading microbes can be studied by quantifying the amount of catabolic genes carried by these microbes. Because the change in the amount of the functional marker genes is relational to the change in cell numbers (Park and Crowley, 2006; Johnsen et al., 2007).The extent of crude oil degradation varied over a wide range (1- 87%) among the isolates. Isolates were predominantly Gram-positive bacteria (79% of total isolates) belonging to the general Bacillus (93%) and Paenibacillus (7%). Among the few gram negative isolates were from the genera Acintobacter, Alcaligenes, Klebsiella, Burkholderia, Pseudomonas, and Williamsia (Obuekwe et al., 2009) , all the 44 isolates obtained from the primary screening on mineral agar also degraded crude oil in liquid culture. However, they varied widely in their ability to degrade crude oil (10- 88%) as the sole carbon and energy source, also B. ceresus degraded the highest amount (72.8%) as the sole carbon and energy source , members of Bacillus spp. constituted the dominant group.

Autochthonous microorgansism is sediments also possessed satisfactory PAH degradation capability and all three PAH were completely degraded after 4 weeks of growth (Yu et al., 2005b).

86 Results and Discussion

Table (2): Growth and extracellular protein of different indigenous bacterial communities on 500 mg/L naphthalene.

Zero time After 7days After 15days After 21days After 28days Bacterial communities O.D Protein O.D Protein O.D Protein O.D Protein O.D Protein (Sample No.) I/Io I/Io I/Io I/Io (Io) ug/ml (I) ug/ml (I) ug/ml (I) ug/ml (I) ug/ml

1 0.148 200.0 0.751 5.0 540.0 1.530 10.3 400.8 1.406 9.5 220.0 1.282 8.6 100.0

2 0.235 19.0 0.981 4.1 210.0 1.600 6.8 200.0 1.240 5.2 120.0 0.880 3.7 80.0

3 0.641 43.8 0.858 1.3 80.0 1.590 2.4 8.4 1.895 2.9 7.6 2.200 3.4 3.8

4 0.910 142.3 1.450 1.6 238.4 1.300 1.4 76.9 1.194 1.3 69.2 1.089 1.1 30.7

5 0.513 57.6 0.677 1.3 138.4 0.881 1.7 30.7 1.151 2.2 15.3 1.421 2.8 7.6

6 0.978 16.9 1.240 1.3 63.0 1.262 1.2 7.6 0.995 1.0 3.8 0.728 0.7 5.6

7 1.020 95.0 1.309 1.3 206.8 1.697 1.6 160.0 2.088 2.0 90.0 2.480 2.4 49.0

87 Results and Discussion

3 S = Sample No. s1 s2 s3 2.5 s4 s5 s6 s7 2 ) nm

60 1.5 . ( D . O 1

0.5

0 0 7 15 21 28 Incubation period (days)

Figure (15): Growth of different indigenous bacterial communities on 500mg/l naphthalene.

600 S = Sample No.

s1 s2 s3 500 s4 s5 s6 s7 400

300

Protein(ug/ml) 200

100

0 0 7 15 21 28 Incubation period (days)

Figure (16): Concentration of extracellular protein of different bacterial communities communities on 500mg/l naphthalene.

88 Results and Discussion

Table (3): Growth and extracellular protein of different indigenous bacterial communities on 250 mg/l phenanthrene.

Zero time After 7days After 15days After 21days After 28days Bacterial O.D communities O.D Protein O.D Protein O.D Protein Protein O.D Protein (Sample No.) I/Io I/Io (I) I/Io I/Io (Io) ug/ml (I) ug/ml (I) ug/ml ug/ml (I) ug/ml

1 0.142 154.0 0.398 2.8 360.0 0.388 2.7 183.0 0.651 4.6 70.7 0.914 6.4 23.0

2 0.253 50.9 0.385 1.5 28.8 0.393 1.6 22.0 0.681 2.7 23.0 0.97 3.8 14.0

3 0.320 49.0 0.556 1.7 77.9 0.477 1.5 60.0 0.399 1.2 44.0 0.304 1.0 12.0

4 0.910 59.7 1.577 1.7 34.0 1.722 1.9 6.0 1.868 2.0 6.0 1.888 2.0 3.8

5 0.723 40.0 0.535 0.7 21.9 0.652 0.9 12.7 0.513 0.7 10.0 0.374 0.5 5.0

6 0.848 44.9 0.664 0.8 23.6 0.770 0.9 13.9 0.645 0.7 12.0 0.520 0.6 4.9

7 1.039 100.8 2.096 2.0 218.0 2.500 2.4 170.7 2.500 2.5 100.0 2.500 2.4 30.0

89 Results and Discussion

3 S = Sample No. 2.5 s1 s2

) 2 s3 nm 1.5 s4 600

.( s5 D .

O 1 s6 s7 0.5

0 0 7 15 21 28 Incubation period (days)

Figure (17): Growth of different indigenous bacterial communities on 250mg/l phenanthrene.

400

350 S = Sample No.

300 s1

s2 250 s3

200 s4 s5 150

Protein (ug/ml) s6

100 s7

50

0 0 7 15 21 28 Incubation period (days)

Figure (18): Concentration of extracellular protein of different indigenous bacterial communities on 250mg/l phenanthrene.

90 Results and Discussion

The same behavior was recorded for community (2), but with less growth to reach 3.8 times the initial at the end of incubation period. The maximum extracellular potein secreted by community (1) was at 7 days incubation and decline as the incubation period increased as shown in Figure (18).

Ability of community (1) to growth on 50 mg/L anthracene have been indicated in Table (4) and Figure (19). As in case of Naph. and Phen. growth of community (1) was the best after 7 and 15 days, but as the incubation period increased more (21 and 28 days) community (2) was found to be superior.

Community (1) secreted the highest extracellular protein 3.0 times the intial after 7 days incubation as indicated in Figure (20).

The growth behavior of community (1) revealed that this community continue the increase in growth as the incubation period increased on 100 mg/L Acen. as indicated in Table (5) and Figure (21). The Same behavior had been recorded for community (2) but with less growth along all the incubation period. The best community in secreting extracellular protein was community (1) as shown in Table (5) and Figure (22). The increase in protein was 5.1 and 5.5 times the intial after 7 and 15 days incubation respectively.

91 Results and Discussion

Table (4): Growth and extracellular protein of different indigenous bacterial communities on 50 mg/l anthracene.

Zero time After 7days After 15days After 21days After 28days Bacterial communities O.D Protein O.D Protein O.D Protein O.D Protein O.D Protein (Sample No.) I/Io I/Io I/Io I/Io (Io) (ug/ml) (I) (ug/ml) (I) (ug/ml) (I) (ug/ml) (I) (ug/ml)

1 0.191 39.3 0.466 2.4 120.4 0.419 2.2 110.0 0.451 2.3 50.7 0.484 2.5 22.0

2 0.254 47.5 0.509 2.0 77.0 0.428 1.7 33.0 0.753 2.9 27.0 1.078 4.2 12.0

3 0.304 50.0 0.419 1.3 66.0 0.480 1.6 8.4 0.338 1.1 7.6 0.196 0.6 3.8

4 0.881 130.0 1.217 1.3 219.0 1.604 1.8 96.7 1.778 2.0 59.0 1.953 2.2 25.5

5 0.922 88.7 1.032 1.1 170.0 1.376 1.5 33.4 1.037 1.1 18.3 0.698 0.7 3.6

6 0.727 23.0 0.783 1.1 48.0 0.938 1.3 8.9 0.751 1.0 7.8 0.725 1.0 7.0

7 1.039 55.8 1.112 1.1 170.0 0.986 0.9 44.0 0.987 0.9 15.3 1.009 1.0 7.0

92 Results and Discussion

2.5 S = Sample No. 2 s1

) s2 1.5 s3 nm s4 600

. ( . s5 D

. 1

O s6 s7 0.5

0 0 7 15 21 28 Incubation period (days)

Figure (19): Growth of different indigenous bacterial communities on 50mg/l anthracene.

250

S = Sample No. 200 s1 s2 150 s3 s4 s5 100 s6 Protein (ug/ml)Protein s7

50

0 0 7 15 21 28 Incubation period (days)

Figure (20): Concentration of extracellular protein of different indigenous bacterial communities on 50mg/l anthracene.

93 Results and Discussion

Table (5): Growth and extracellular protein of different indigenous bacterial communities on 100 mg/l acenaphthene.

Bacterial Zero time After 7days After 15days After 21days After 28days communities (Sample No.) O.D Protein O.D Protein O.D Protein O.D Protein O.D Protein I/Io I/Io I/Io I/Io (Io) ug/ml (I) ug/ml (I) ug/ml (I) ug/ml (I) ug/ml

1 0.131 50.0 0.376 2.9 260.0 0.407 3.1 280.0 0.612 4.7 120.0 0.817 6.2 50.0

2 0.208 50.6 0.502 2.4 120.3 0.699 3.3 130.0 0.724 3.4 80.6 0.749 3.6 30.7

3 0.350 41.2 0.384 1.0 83.6 0.325 0.9 29.0 0.308 0.9 18.0 0.291 0.8 11.6

4 0.444 22.8 0.588 1.3 40.3 0.942 2.1 19.4 0.743 1.7 21.8 0.545 1.2 10.0

5 0.669 89.0 0.536 0.8 160.0 0.495 0.7 34.7 0.445 0.7 18.9 0.396 0.6 5.0

6 0.827 60.0 0.922 1.1 70.7 2.500 3.0 59.0 2.182 2.6 44.0 1.864 2.2 12.0

7 1.039 30.0 1.856 1.7 80.0 1.637 1.6 21.0 1.275 1.2 12.0 0.913 0.9 10.7

94 Results and Discussion

3

2.5 S = Sample No.

2 s1 ) s2 nm s3

600 1.5 .( D

. s4 O 1 s5 s6 0.5 s7

0 0 7 15 21 28 Incubation period (days)

Figure (21): Growth of different indigenous bacterial communities on 100mg/l acenaphthene.

300

250 S = Sample No.

s1 200 s2 s3 s4 150 s5 s6 Protein Protein (ug/ml) 100 s7

50

0 0 7 15 21 28 Incubation period (days)

Figure (22): Concentration of extracellular protein of different indigenous bacterial communities on 100mg/l acenaphthene.

95 Results and Discussion

However, the best communities in growth on 10.0 mg/L fluoranthene a log all the incubation period were communities (2), (4) and (7) as indicated in Table (6) and Figure (23). The highest growth 2.9, 2.2 and 2.4 times the initial for communites (2), (4) and (7) respectively were recorded after 28 days.

The best community in secreating extracellular protein was community (4). Secretion of protein was 7.1 and 8.2 times the initial after 7 and 15 days respectively. The same trend have been recorded but in less protein secretion in case of community (2). The highest extracellular potein (5.8 times) was found in case of community (7) after 7 days incubation as shown in figure (24).

Grwoth of communities (1) and (2) on 100g/L pyrene was the best a long the whole of incubation period as indicated in Table (7) and Figure (25). The results revealed that both growth of communities (1) and (2) continue the increase as the incubation period increased to reach the highest growth at the end of incubation period to be 4.1 and 3.1 times the intial respectively.

The maximum extracellular protein secreted by communites (1) and (2) when grown on Pyr. was 2.25 and 1.8 times the intial after 7 days incubation, then began to decrease as the incubation increased as cleared in Figure (26).

96 Results and Discussion

Table (6): Growth and extracellular protein of different indigenous bacterial communities on 10 mg/l fluoranthene.

Zero time After 7days After 15days After 21days After 28days Bacterial communities O.D Protein O.D Protein O.D Protein O.D Protein O.D Protein (Sample No.) I/Io I/Io I/Io I/Io (Io) (ug/ml) (I) (ug/ml) (I) (ug/ml) (I) (ug/ml) (I) (ug/ml)

1 0.255 30.0 0.463 1.8 78.0 0.477 1.9 50.8 0.509 2.0 26.0 0.541 2.1 18.0

2 0.226 46.0 0.462 2.0 87.0 0.436 1.9 120.0 0.549 2.4 80.0.0 0.663 2.9 30.0

3 0.776 55.9 0.862 1.1 60.0 0.919 1.9 30.0 0.781 1.0 13.0 0.644 0.8 7.0

4 0.998 18.0 2.22 2.2 129.6 2.500 2.5 149.0.8 2.352 2.4 30.0 2.205 2.2 12.0

5 0.550 34.7 0.672 1.2 50.8 1.193 2.2 37.0 0.996 1.8 16.0 0.800 1.4 4.0

6 0.747 68.4 0.781 1.0 70.0 1.306 1.7 37.0 0.990 1.3 12.8 0.675 0.9 12.0

7 1.039 34.0 1.895 1.8 200.0 2.500 2.4 180.0 2.500 2.4 39.0 2.500 2.4 39.0

97 Results and Discussion

3 S = Sample No.

2.5 s1 s2 2 ) s3

nm s4 1.5 600 s5 . ( D

. s6 O 1 s7

0.5

0 0 7 15 21 28 Incubation period (days)

Figure (23): Growth of different indigenous bacterial communities on 10mg/l fluoranthene.

250

200 S = Sample No. s1 s2 150 s3 s4 s5 100 s6

Protein (ug/ml)Protein s7

50

0 0 7 15 21 28 Incubation period (days)

Figure (24): Concentration of extracellular protein of different indigenous bacterial communities on 10mg/l fluoranthene.

98 Results and Discussion

Table (7): Growth and extracellular protein of different indigenous bacterial communities on 100 ug/l pyrene.

Bacterial Zero time After 7days After 15days After 21 days After 28days communities O.D Protein O.D Protein O.D Protein O Protein O.D Protein (Sample No.) I/Io I/Io I/Io I/Io (Io) (ug/ml) (I) (ug/ml) (I) (ug/ml) D(I) (ug/ml) (I) (ug/ml)

1 0.168 120.0 0.409 2.4 270.0 0.394 2.3 110.0 0.541 3.2 80.9 0.689 4.1 44.0

2 0.231 160.9 0.472 2.0 290.0 0.463 2.0 130.9 0.594 2.6 60.9 0.726 3.1 44.0

3 0.384 80.0 0.457 1.2 100.0 0.721 1.9 64.0 0.485 1.3 30.0 0.250 0.7 10.0

4 0.564 60.7 0.632 1.1 79.0 0.885 1.6 40.0 0.942 1.7 33.0 1.00 1.8 30.0

5 0.883 49.8 0.985 1.1 70.0 2.500 2.8 99.0 1.705 1.9 60.0 0.911 1.0 40.0

6 0.882 60.8 0.969 1.0 80.0 2.500 2.8 160.0 1.700 1.9 70.0 0.901 1.0 50.8

7 0.374 59.7 0.470 1.6 210.8 2.500 6.6 270.0 1.725 4.6 210.0 0.950 2.5 100.8

99 Results and Discussion

3

2.5 S = Sample No.

s1 2 ) s2 nm s3 1.5 600

. ( s4 D . O 1 s5 s6

0.5 s7

0 0 7 15 21 28 Incubation period (days)

Figure (25): Growth of different indigenous bacterial communities on 100ug/l pyrene.

350

300 S = Sample No. 250 s1 s2 s3 200 s4 s5 150 s6

Protein (ug/ml) s7 100

50

0 0 7 15 21 28 Incubation period (days)

Figure (26): Concentration of extracellular protein of different indigenous bacterial communities on 100 ug/l pyrene.

100 Results and Discussion

Growth behavior of communities (1) and (2) on 100 g/L benzo-a- anthracene revealed that, as the incubation period increased, the growth increased to reach the maximum growth 3.0 and 4.0 times the initial after 28 days respectively as indicated in Table (8) and Figure (27). Surprisingly, community (7) reached the maximum growth (1.5 times) in a shorter incubation period (15 days) and began to decrease. The maximum extracellular protein for communities (1) and (2) had been recorded after 7 days incubation period to be 2.33 and 3.67 times the initial respectively, as indicated in Figure (28).

The count of growth at the end of incubation period (28 days) as indicated in Table (9), (10) and Fig (29) revealed that the best community in growth on the seven PAHs was community (1). The intial count of community (1) was 6.0 x105 CFU/ml. The highest count of community (1), 3.4 x107 CFU/ml was recorded on Pyrene. Also, the count of community (1) on Anth., Ace, Phen increased than that of intial count to be 1.2x 107, 7.0x106 and 4.0x106 CFU/ml respectively. The results also revealed that, communities (1) and (2) and (3) were the best communities growing on Phen. and Anth. as a sole carbon and energy source even at the end of incubation period when the growth began to decline.

101 Results and Discussion

Table (8): Growth and extracellular protein of different indigenous bacterial communities on 100 ug/l benzo-a-anthracene.

Bacterial Zero time After 7days After 15days After 21 days After 28days communities O.D Protein O.D Protein O.D Protein O.D Protein O.D Protein (Sample No.) I/Io I/Io I/Io I/Io (Io) (ug/ml) (I) (ug/ml) (I) (ug/ml) (I) (ug/ml) (I) (ug/ml)

1 0.135 90.0 0.287 2.1 210.0 0.394 2.9 130.9 0.402 3.0 60.9 0.411 3.0 39.8

2 0.211 85.9 0.371 1.8 316.0 0.620 2.9 160.0 0.741 3.5 50.8 0.863 4.0 40.8

3 0.627 60.8 0.973 1.6 60.0 1.269 0.4 30.9 0.981 1.6 29.9 0.693 1.1 17.9

4 1.378 42.0 1.509 1.0 30.9 1.613 1.2 23.8 1.662 1.2 16.0 1.712 1.2 10.9

5 1.447 40.0 1.547 1.0 39.0 1.912 1.3 23.6 1.702 1.2 12.0 1.492 1.0 11.0

6 0.561 36.0 0.656 1.2 39.0 0.987 1.8 13.0 1.111 2.0 7.0 1.235 2.2 8.0

7 1.135 39.0 1.382 1.2 20.0 1.690 1.5 22.0 1.595 1.4 12.0 1.500 1.3 6.0

102 Results and Discussion

2.5

2 S = Sample No.

s1

) s2 1.5

nm s3

600 s4 . ( . D

. 1 s5 O s6 s7 0.5

0 0 7 15 21 28 Incubation period (days)

Figure (27): Growth of different indigenous bacterial communities on 100ug/l benzo-a-anthracene.

350

300 S = Sample No.

s1 250 s2 s3 200 s4

150 s5 s6 Protein (ug/ml)Protein 100 s7

50

0 0 7 15 21 28 Incubation period (days)

Figure (28): Concentration of extracellular protein of different indigenous bacterial communities on 100ug/l benzo-a-anthracene.

103 Results and Discussion

Also, from the previous results it was clear that community (1) of contaminated soil at the surface with chronic exposure to pollution with crude oil at the same place of contamination (zero meter) followed by community (2) with the same characters as community (1) but differ in recent exposure for contamination having the highest abilities to grow and utilize PAHs as a sole carbon and energy source. The two communities represent the surface i.e., the diversity of bacterial communities of surface soil contain more efficient bacterial strains than that found in deep soils.

From all the above results, it is clear that communities (1) and (2) were the most efficient communites in growing on different PAHs.

All the previous results of this study could be confirmed and explained by different studies of other investigators as the following.

104 Results and Discussion

Table (9): Count of different indigenous bacterial communities on different polycyclic aromatic hydrocarbons (PAHs) after 28 days incubation. Initial count Indigenous bacterial communities counts (CFU/ml)

No. of rings 1 2 3 4 5 6 7

Compounds 6.0*105 8.0*105 4.0*106 1.6*107 4.0*106 1.0*106 8.0*105 5 4 6 7 6 6 5 2 Naphthalene 8.0*10 4.0*10 1.0* 10 1.6* 10 4.0* 10 1.0*10 8.0*10 6 7 7 7 6 5 6 3 Phenanthrene 4.0*10 2.8*10 1.1*10 1.6*10 6.0*10 6.0*10 2.0*10 7 5 7 6 6 6 6 3 Anthracene 1.2*10 9.0*10 1.2*10 5.0*10 4.0*10 2.0*10 4.0*10

6 6 6 6 6 5 6 3 Acenaphthene 7.0*10 2.8*10 4.0*10 8.0*10 4.0*10 6.0*10 3.0*10

5 4 6 7 6 5 6 4 Fluoranthene 4.0*10 2.0*10 9.0*10 1.9*10 5.0*10 3.0*10 1.0*10 7 5 6 6 6 5 6 4 Pyrene 3.4*10 4.0*10 8.0*10 8.0*10 2.0*10 4.0*10 6.0*10

4 B(a) anthracene 6.0*104 7.0*104 4.6*107 7.0*106 4.0*106 8.0*105 1.2*106

105 Results and Discussion

Table (10): Increase of indigenous bacterial communities count after 28 days of incubation period on different PAHs.

Log indigenous bacterial communities count 1 2 3 4 5 6 7 compounds Log LogI1/ Log LogI2/ Log LogI3/ Log LogI4/ Log LogI5/ Log LogI6/ Log LogI7/ I 1 LogIo I 2 LogIo I 3 LogIo I 4 LogIo I 5 LogIo I 6 LogIo I 7 LogIo

naphthalene 5.987 1.0 4.873 0.8 6.0 0.9 7.204 1.0 6.602 1.0 6.000 1.0 5.903 1.0 phenanthrene 6.602 1.1 7.447 1.3 7.041 1.0 7.204 1.0 6.778 1.0 5.778 0.9 6.301 1.0

Anthracene 7.079 1.2 5.954 1.0 7.079 1.0 6.698 0.9 6.602 1.0 6.301 1.0 6.602 1.1 acenaphthene 6.845 1.2 6.447 1.0 6.602 1.0 6.903 0.9 6.602 1.0 5.778 0.9 6.477 1.0 fluoranthene 5.602 1.0 4.301 0.7 6.954 1.0 7.278 1.0 6.698 1.0 5.477 0.9 6.000 1.0

pyrene 7.531 1.3 5.602 0.9 6.903 1.0 6.903 0.9 6.301 0.9 5.602 0.9 6.778 1.1

Benzo (a) 3.647 0.6 5.728 0.9 7.662 1.2 6.845 0.9 6.602 1.0 5.903 1.0 6.079 1.0 anthracene

106 Results and Discussion

Naphthalene Phenanthrene Anthracene Acenaphthene Fluoranthene Pyrene B-a-anthracene

8 7 6 5 4

log log N 3 2 1 0 1 2 3 4 5 6 7 Indigenous bacterial communities

Figure (29): Count of different indigenous bacterial communities on different polycyclic aromatic hydrocarbons (PAHs) after 28 days incubation.

107 Results and Discussion

Polycyclic aromatic hydrocarbon degradation was directly related to the historical environmental pollution of the sampling sites examined, the length of biodegradation assessment, temperature, and the molecular size of the polyclic aromatic hydrocarbon substrate (Sherrill and Sayler, 1980).

Soil enzyme activities are the driving force behind all the biochemical transformation occurring in oil. Their revaluation may provide useful information on soil microbial activity and be helpful to establish effects of soil specific environmental conditions (Dick et al., 1996).

Phelps and Young (1999) found that in the case of contamination by fuel hydrocarbons, it is now well known that many microorganisms indigenous to soil can oxidize (mineralize) the contaminants to harmless carbon dioxide and water.

Bioremediation of polycyclic aromatic hydrocarbon (PAH)- polluted soil is severely hampered by the low rate of degradation of the higher PAH, particularly the four-and five-ring PAH. These higher PAH have very low water solubility and are often tightly bound to soil particles (Wilson and Jones, 1993).

The duration of the acclimation period depends on a number of environmental variables such as contaminant concentration, bioavailability, pH, temperature, levels of nitrogen and present, aeration status, and prior exposure the microbial communities to PAHs (Alexander, 1999). Heterogenous distribution of PAHs in soil combined with their absorption to organic matter and low levels of diffusibility, limits bacterial access to PAHs as substrates (Johnsen et al., 2005). Many bacteria produce biosurfactants when grown on hydrocarbons and these can increase PAH solubility (Van Dyke et al., 1993).

Huesemann et al. (2001) have demosntrated that in six aged soil samples for most of the 2- and 3- ring PAHs, the degrdations were governed

108 Results and Discussion by the desorption from soils, while for the higher molecular-weight PAHs microbial degradation potential was the controlling factor.

Biodegradation was not significantly influenced by the addition of such carbon sources as , pyruvate, and yeast extract, but was significantly influenced by the addition of , sulfate, and phosphate results show that anthracene, fluroene, and pyrene biodegrdation was enhanced by the presence of phenanthrene, but that phenanthrene treatment did not induce benzo(a) pyrene biodegradation during a 12-day incubation period (Yuan et al., 2001).

Cole et al. (2000) reported that no appreciable changes in fluoranthene concentrations in spiked sandy sediments stored over a period of 170 d at 4C.

Chang et al. (2003) demonstrated that no significant differences were found in PAH degradation rates within a pH range of 6.0-8.0, but a delay was noted at pH 9.0.

However, efficiency of naturally occurring microorganisms for field bioremediation could be significantly improved by optimizing certain factors such as bioavailability, adsorption and mass transfer. could also have an important role in enhancing biodegrdation of pollutants (Samanta et al., 2002 and Feitkenhauer et al., 2003).

Pizzul et al. (2007) revealed that low bioavailability, toxicity, complex and diverse structural configuration, electrochemical stability, low hydrophobic nature, strong sorption phenomena of PAHs and non-uniform spatial distribution of microorganisms in the soil matrix makes bioremediation of PAHs extremely complex.

MacNaughton et al. (1999) demonstrated a microbial community shift and a dominant growth of Gram-negative microorganisms in a crude oil- contaminated costal site.

109 Results and Discussion

Two bacteria strains Sphingomonas sp. Strain ZP1 and Tistrella sp. Strain, ZP5 were identified as phenanthrene-degrading ones, based on Gram staining, oxidase reaction, biochemical tests, FAME analysis, G+C content and 16S rDNA gene sequence analysis. Zhao et al., 2008 isolated these two bacteria strains Sphingomonas sp. ZP1 and Tistrella sp. ZP5 from soil samples contaminated with polyclic aromatic hydrocarbon (PAH)- containing waste from field in Shanghai, China. Strain Sphingomonas sp. ZP1 was able to degrade naphthalene, phenanthrene, toluene, methanol and , salicylic acid and Tween 80. Moreover, it can remove nearly all the phenanthrene at 0.025% concentration in 8 days (Zhao et al., 2008).

The microbial activity in soils was a critical factor governing the degradation of organic micro-pollutants. The microbial activities were relatively lower in the soils with the lowest and highest organic matter content, which were likely due to the nutrition limit and PAH sequestration. The nutrition support and sequestration were the two major mechanisms, that solid organic matter influenced the development of microbial PAHs degradation potentials (Yang et al., 2011).

In very low oxygen environemtns, there may be microbial degradation of LMW PAH via non-oxygen dependent mechanisms (Heider et al., 1999).

Three- and four-ring PAHs could be degraded by the indegenous microbial community under aerobic conditions, but anaerobic metabolism based on iron and sulphate reduction was not coupled with PAH degradation of even the simples 3-ring compounds like phenanthrene. addition stimulated both aerobic and anaerobic respiration, but had no effect on PAH dissipation. We conclude that natural attenuation of PAHs in polluted river sediments under anaerobic conditions is exceedingly slow (Quantin et al., 2005). PAH degradation is most extensive under aerobic conditions, but is known to occur to some extent in anaerobic environments (Davidova et al.,

110 Results and Discussion

2007). and sulphate have been reported as alternative electron acceptors (Heider et al., 1998).

Hatzinger and Alexander (1997) demonstrated that the extent of mineralization and the rate of phenanthrene biodegrdation declined with increasing contact time between phenathrene and the soil. Tang et al. (1998) also reported that aging decreased the amont of phenanthrene, anthracene, fluoranthene, and pyrene available to the microorganism to enhance biodegradation, researchers have used surfactants (Yeom et al., 1996) and organic solvents (Kilbane, 1997) to improve the availability of PAHs.

Bacterial communities of phenanthrene-degraders were present in a higher density in the aggregates corresponding to sand (2000-50 mm) and clay (<2 mm). Chemical analysis show that remaining PAHs (low and high molecular weight) were much more concentrated in the fine soil fractions (fine silt and clay) and were present at a very low content in the larger aggregate size fractions. Differences in amounts of solubilized phenanthrene between sand and clay aggregate size fractions would be related to differences in adsorption capacities of phenanthrene by clay and sand aggregates (Amellal et al., 2001).

PAH degradation is generally an aerobic process, although anaerobic degradation has been reported (Bianchin et al., 2006) Oxygen levels in PAH contaminated environments such as soils and sediments are typically well below the levels required for aerobic transformation of PAHs. Increasing that increase the interparticle space within the soil matrix, direct injection of oxygen (Sparging), or introduction of oxygen generating species such as

H2O2 have been shown to increase both the rate and often the extent of PAH degradation (Kaplan and Kitts, 2004).

The rate of PAH degradation is generally faster in soil that has a history of PAH contamination. A study by Johnsen and Karlson in (2005) revealed that the rate of 14C-labeled pyrene and phenanthrene mineralization was inversely proportional to the PAH content of 13 different soils PAH 111 Results and Discussion degradation in environments with no history of previous contamination may result from exposure to PAHs from natural sources such as biogenic synthesis (Grimalt et al., 2004), Atomospheric deposition of PAHs from contaminated sites, or the presence of microorganisms that produce broad specificity enzymes such as laccases.

Successful soil augmentation requires not only knowledge on type and level of contaminants but also suitable strains of microorganisms or their consortia. The selection of proper culture should take into consideration the following features of microorganisms: fast growth, easy cultured, to withstand high concentrations of contaminants and the ability to survive in a wide range of environmental conditions. Particularly attractive are ‘heirloom’ microorgnaisms that are maintained and handed down for many years and are specifically modified for bioaugmentation purpose (Singer et al., 2005). For remediation of sites contaminated with various PAHs and biphenyls it is necessary to use strains able to produce surfactants to make these pollutants more accessible (Gentry et al., 2004).

In situ horizontal transfer of PAH metabolizing genes (nahAc and phnAC) has been reported between phylogenetically distinct bacteria, and this may also explain the PAH degradative capacity of communities with no known exposure to PAHs (Park and Crowley, 2006). Previous exposure of a microbial community to one PAH has frequently been reported to reduce the acclimation period for degradation of other PAHs (Beckles et al., 1997).

The high percentage of Bacillus strains characterized in our work (66.6%) should be related with the property of these microorganisms to colonize environments contaminated with hydrocarbons (Zhuang et al., 2002). In this context, Ijah and Antai reported that Bacillus species were the predominant microorganisms in highly polluted soil samples (Ijah and Antai, 2003).

112 Results and Discussion

Biostimulation with addition of mineral salt medium degraded over 97% of all three PAHs, showing that amendment could enhance pyrene degradation (Yu et al., 2005b).

However, both cis- and trans-didhyrodiols can be dehydrated to form the 9-hydrodiols can be dehydrated to form the 9-hydroxyphenanthrene. Moreover, the consortium might also transform phenanthrene to mon- hydroxyl phenanthrene via mono-oxygenase. Therefore, the identity of other metabolites cannot be confirmed. On the one hand, a novel metabolite, trihydroxy phenanthrene was detected. The insertion of three hydroxyl groups might be produced by enzymatic reactions of both mono- and di-oxygenase systems. These results showed that the bacterial consortium has a complicated enzymatic profile for degradation, suggesting why it has a higher degradation capability to degrade PAHs rather than pure isolate (Luan et al., 2006).

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) in soil is mainly performed by endogenous bacteria (Corgié et al., 2006).

Nievas et al. (2008) suggested that the microbial consortium used hydrocarbons and yeast extract as carbon source.

Any factor stimulating the growth of degrading populations (e.g., addition of , aeration, etc.) would thus affect degradation rates. For example, addition of phenathrene increased the initial rate of fluoranthene degradation in soil, but a similar effect was observed if a biosurfactant was added instead of the 3-ring PAH (Hickey et al., 2007).

Zhang et al. (2008) observed that soil pH was discovered to affect the process whereby the highest pyrene and benzo(a)pyrene degradation rates were obtained at acidic conditions, while phenathrene was most significantly degraded at alkaline conditions. Additionally, the presence of humic acid in soil was found to enhance PAH photocatalytic degradation by sensitizing radicals capable of oxidizing PAHs.

113 Results and Discussion

Among biotic factors the most important is the selection of proper microorganisms that can not only degrade contaminants but can also successfully compete with indigenous miroflora (Mrozik and Piotrowskaseget, 2010).

Towell et al. (2011) indicated that hydrocarbon mineralization by the indigenous microbial community was monitored over 23d. hydrocarbon mineralization enhancement by nutrient amendment (Biostimulation), hydrocarbon degrader addition (Bioaugmentation) and combined nutrient and degrader amendment. In general, the rates and extents of mineralization will be dependent upon treatment type, nature of the contamination and adaptation of the ingenous microbial community.

3.2. Determination of the total bacterial count and the hydrocarbon degrading bacteria (HDB) found in each community

Growth of the seven different communities on L.B medium to give the total bacterial count and BSM amended with 1% crude oil to determined the count of hydrocarbon degrading bacteria (HDB) revealed that community (4) was the highest growth (1.6x107 CFU/ml) as total bacterial count, while communities (3) was the highest (3.0x105 CFU/ml) as hydrocarbon degrading bacteria as indicated in Table (11) and Fig. (30). Although, the best count in hydrocarbon degrading bacteria was in communities (3) , communities (1), (2) were the best, in growing and degrading PAHs.

This may be explained on the bases that this count was on the whole crude oil not on specific PAHs as a substrate. The second reason for these results, may be the efficiency of the individual strain in the community not on their count as a number.

114 Results and Discussion

Table (11): Count of different indigenous bacterial communities on different media.

% Log No. of Bacterial Total bacterial H D B count N HDB communities count (CFU/ml) logN logN (CFU/ml) Log N (sample No.) (Io) TBC

1 6.0 *105 5.778 1.0 *10 4 4.000 69.2 2 8.0 *10 5 5.903 2.0 *10 4 4.301 72.8 3 4.0 *10 6 6.602 3.0* 10 5 5.477 82.9 4 1.6 *10 7 7.204 2.0* 10 5 5.301 73.5 5 4.0 *10 6 6.602 1.6* 10 3 4.200 63.6 6 1.0*10 6 6.000 1.0* 10 5 5.000 83.3 7 8.0 *10 5 5.903 1.0* 10 3 3.000 50.8

*HDB=hydrocarbon degrading bacteria. **TBC=Total bacterial count on L.B. medium.

Total bacterial count (CFU/ml) H D B count of (CFU/ml)

8

7

6

5

4 Log N 3

2

1

0 1 2 3 4 5 6 7 Indigenous bacterial communities

Figure (30): Count of different indigenous bacterial communities on different media. 115 Results and Discussion

Bioremediation of areas contaminated with crude, fuel or other hydrocarbon compounds is feasible due to their biodegradability and the diversity of degrading microorganisms present in these sites. Hydrocarbon degrading bacteria are widely spread in polluted soil and water, and research has shown that application of hydrocarbon increases the number of bacteria (Zhuang et al., 2002).

3.3. Isolation and determination of the most potent strains having the ability to degrade different PAHs

The ability of different indigenous isolated stains to grow on different concentrations of PAHs had been indicated in Table (12). From the results of Table (12), it is clear that isolates of code MAM-26, MAM-29, MAM-43, MAM-62, MAM-68 and MAM-78 are the best isolates having the abilities to grow on different PAHs as a sole carbon and energy source. These six most potent strains were used for further studies by using each isolate with different concentrations of different PAHs. Characterization of these six isolates have been indicated in Table (13).

The degradation test of phenanthrene, anthracene and pyrene was carried out at 30C, pH 7.0.The degradation rate of Janibacter anophelis strain JY 11 decreased when the initial concentration of each kind of PAHs is low (100 ppm) or high (1000 ppm). This is mainly because a lower PAHs concentration is not enough for supporting the growth of J.anophelis strain JY11. While, higher PAHs concentration will lead to increasing of PAHs metabolites’ toxicity (Zhang et al., 2009).

116 Results and Discussion

Table (12): The ability of indigenous isolated strains to grown on different PAHs at different concentrations. Comp. B-a- Pyrene Naphthalene Anthracene Phenanthrene Acenaphthene Fluoranthene Isolate anthracene 150 500 mg/l 100 mg/l 500 mg/l 250 mg/l 20 mg/l code 150 ug/l ug/l MAM -1 + + + + + + ++ MAM -2 + + + -ve ++ + + MAM -3 + + + + + + + MAM-5 ++ -ve ++ + + + + MAM-14 ++++ ++ ++ + ++ +++ +

MAM-15 ++ ++ ++ ++ ++ + ++ MAM-16 + + + -ve + + -ve MAM-17 + ++ +++ -ve ++ + + MAM-18 ++ +++ ++ ++ + + ++ MAM-20 + -ve -ve + -ve -ve -ve MAM-21 ++ ++ + ++ +++ +++ +++ MAM-22 -ve -ve -ve -ve + -ve + MAM-23 + + ++ + ++ + + MAM-25 ++ +++ ++ + ++ ++ ++ MAM-26 ++++ +++ +++ ++++ +++ ++++ ++++ MAM-27 + +++ + + +++ +++ +++ MAM-28 +++ +++ ++ +++ +++ ++ +++ MAM-29 +++ ++++ +++ ++++ ++++ +++ ++++ MAM-30 -ve + ++ ++ + + + MAM-31 + + ++ ++ +++ +++ ++ MAM-32 + ++ + + + + -ve MAM-34 + -ve + -ve + + +

117 Results and Discussion

Continue of Table (12):

Comp. B-a- Pyrene Naphthalene Anthracene Phenanthrene Acenaphthene Fluoranthene Isolate anthracene 150 500 mg/l 100 mg/l 500 mg/l 250 mg/l 20 mg/l code 150 ug/l ug/l MAM-35 +++ ++ + ++ ++ ++ +++ MAM-37 +++ ++ + + +++ +++ ++ MAM-38 ++ ++ +++ ++ ++ +++ +++ MAM-40 ++ +++ ++ + +++ +++ ++ MAM-41 + ++ +++ -ve +++ ++++ +++ MAM-43 ++++ ++++ ++++ +++ ++++ +++ ++++ MAM-44 +++ + ++ + ++ +++ + MAM-47 +++ +++ + +++ ++++ +++ +++ MAM-49 ++ ++ + ++ +++ + ++ MAM-50 +++ -ve + -ve -ve ++ -ve MAM-52 ++ +++ +++ ++ +++ ++ +++ MAM-53 ++ ++ + + ++ ++ + MAM-54 +++ +++ ++ +++ +++ +++ +++ MAM-57 ++ +++ ++ +++ +++ +++ ++ MAM-58 + -ve -ve + -ve -ve -ve MAM-59 ++ ++ ++ ++ ++ ++ +++ MAM-62 ++++ +++ ++++ +++ ++++ ++++ ++++ MAM-64 +++ ++ ++ +++ +++ ++ ++ MAM-66 + -ve -ve + + + -ve MAM-68 ++++ ++++ ++++ ++++ ++++ ++++ ++++ MAM-69 + -ve + ++++ + + -ve MAM-78 ++++ ++++ +++ ++++ +++++ ++++ ++++

118 Results and Discussion

Table (13): Characters of isolated strains on BSM agar supplemented with PAHs compounds.

Sample Isolate code PAHs Characters Gram stain No. MAM-11 phenanthrene Small, circular, concave, shiny. MAM-12 phenanthrene medium, regular, concave, shiny, yellowish. MAM-13 phenanthrene Large, convex, irregular, creamy, shiny. MAM-14 phenanthrene Medium, concave, regular, circular, shiny, yellowish. MAM-15 phenanthrene Medium, regular, concave, creamy. MAM-16 phenanthrene Small, very concave, circular, yellowish-creamy. MAM-17 phenanthrene Yellow , wrinkled, concave, moderate, round MAM-18 phenanthrene Small, round, regular, concave. 1 MAM-19 phenanthrene Round, regular, yellow, concave, medium. MAM-20 phenanthrene Large, flat, creamy, concave, shiny. MAM-21 phenanthrene Medium, creamy, flat, shiny. MAM-22 phenanthrene Medium, irregular, flat, concave, creamy, shiny. MAM-23 phenanthrene Large, flat, shiny, irregular, creamy. MAM-24 phenanthrene Small, round, concave, creamy. MAM-26 Pyrene Medium, concave, shiny, yellow, transparent G+ve long rod Bacilli MAM-27 Pyrene Medium-large, slightly concave, shiny, creamy. MAM-75 Acenaphthene Fungi, white. MAM-1 Anthracene Medium, circular, shiny, flat, white. MAM-2 Anthracene Medium, circular, creamy, slightly concave, shiny. MAM-3 Anthracene medium , Circular, concave , creamy-pink. MAM-4 Anthracene Medium, white. MAM-5 Anthracene Medium, concave, circular, shiny, pink. MAM-6 Anthracene Medium, irregular, flat, shiny, creamy. MAM-7 Anthracene Circular, Light pink. 2 MAM-8 Anthracene Large, irregular, flat, shiny, creamy. MAM-9 Anthracene small- medium, circular, irregular, concave, creamy, shiny. MAM-10 Anthracene Medium, concave, regular, pink. MAM-48 Acenaphthene Medium, concave at center, shiny, pink. MAM-63 Acenaphthene Small-medium , regular, shiny, concave, pink-creamy. MAM-71 Fluoranthene Fungi, white, brown at center. MAM-72 Fluoranthene Fungi, white, brown at center. MAM-73 Fluoranthene Fungi, white, brown at center. 6 MAM-38 B-a-anthracene Medium, wrinkled, pink. MAM-43 Acenaphthene Large, irregular, flat, white, G +ve,long rod spore forming bacilli MAM-49 Anthracene Small, concave, orange. MAM-70 Anthracene Medium, wrinkled, irregular, buff. MAM-28 B-a-anthracene large , Wrinkled, opaque, irregular. MAM-29 B-a-anthracene Small-concave creamy G-ve short rods MAM-30 Anthracene Small-medium, concave, regular, shiny. 7 MAM-39 Anthracene Medium, round, regular, shiny, creamy-pink. MAM-65 Acenaphthene Small, wrinkled at edges (opaque), smooth at center(shiny), flat, regular. MAM-74 B-a-anthracene Fungi, white. MAM-66 Acenaphthene Round, regular, concave, shiny, lemon in color. MAM-67 Acenaphthene Medium, regular, round, pink. 7 MAM-68 Acenaphthene Medium, regular, concave, shiny. G+ve bacilli 9 MAM-32 B-a-anthracene Large, irregular, flat, whitish-creamy.

119 Results and Discussion

Sample Isolate code PAHs Characters Gram stain No. MAM-35 Anthracene Medium, flat, Creamy-pink, shiny, slightly convex at center. MAM-76 Anthracene Very large fungi , white, brown at center. MAM-77 Anthracene Fungi, whitish-yellow. MAM-25 Fluorancene Small, round, yellow. MAM-31 B-a-anthracene Wrinkled, brown , opaque. 11 MAM-59 Fluoranthene Medium, circular, regular, wrinkled, concave, pink. MAM-60 Fluoranthene Large, regular, circular, pink. MAM-61 Fluoranthene Small-medium, very wrinkled, simon. MAM-33 B-a-anthracene Flat, shiny, brown. MAM-34 B-a-anthracene Very large, irregular, shiny, creamy. MAM-36 B-a-anthracene Medium, regular, opaque, creamy. Medium-large, transparent, round regular, shiny, MAM-37 B-a-anthracene whitish-creamy. Medium, regular, concave, shiny. G+ve long MAM-78 B-a-anthracene rod bacilli MAM-40 fluoranthene Large, irregular, flat, shiny, creamy. MAM-41 fluoranthene Small, wrinkled. MAM-42 B-a-anthracene Small, white. MAM-44 B-a-anthracene Medium, regular, brown, brown pigment. MAM-45 B-a-anthracene Large, irregular, shiny, creamy. MAM-46 Anthracene Small-medium, wrinkled, opaque, brown. MAM-47 Acenaphthene Large, irregular, flat, shiny, creamy. 14 MAM-50 Fluoranthene Medium, round, whitish-creamy. MAM-51 Acenaphthene Medium, irregular, concave at center, opaque, lemon in color. MAM-52 Acenaphthene Medium-large, round, regular, flat, wrinkled, simon. MAM-53 Acenaphthene Medium, wrinkled, smooth at edges, regular, round, simon. MAM-54 Acenaphthene Medium, circular, regular, flat, shiny, creamy. MAM-55 Acenaphthene Medium, regular, creamy-pink, yellow. MAM-56 Acenaphthene Medium, convex, simon. MAM-57 Acenaphthene Medium, circular, regular, shiny, concave, pink. MAM-58 Acenaphthene Medium, circular, regular, shiny, concave, pink. MAM-62 Acenaphthene Large, smooth, irregular, simon buff. G +ve long rod bacilli MAM-69 Anthracene Medium, round, flat, shiny, pink. MAM-64 Fluoranthene Small, round, shiny, concave, deep-yellow. MAM-4 G-ve bacilli (E.cloacae)

120 Results and Discussion

3.4. Growth and degradation of naphthalene by the most potent isolated strains Eight strains of Bacillus pumilus, two strains of B. subtilis, three strains of Micrococcus luteus, one strain of Alcaligenes faecalis and one strain of Enterobacter sp. able to grow on mineral liquid media amended with naphthalene, phenanthrene, fluoranthene or pyrene as sole carbon and energy source (Toledo et al. 2006). Fifteen bacterial strains isolated from solid waste oil samples were selected due to their capacity of growing in the presence of hydrocarbons. The isolates were identified by PCR of the 16S rDNA gene using fD1 and rD1 primers. The majority of the strains belonged to general Bacillus, Bacillus pumilus (eight strains) and Bacillus subtilis (two strains). Besides, there strains were identified as Micrococcus luteus, one as Alcaligenes faecalis and one strain as Enterobacter sp. Growth of the above-mentioned strains in mineral liquid media amended with naphthalene, phenanthrene, fluoranthene or pyrene as sole carbon source was studied and our results showed that these strains can tolerate and remove different polycyclic aromatic hydrocarbons that may be toxic in the environment polluted with hydrocarbons. Finally, the capacity of certain strains to emulsify , xilene, toluene, and crude oil, and its ability to remove hydrocarbons, look promising for its application in bioremediation technologies (Toledo et al., 2006). Pseudomonas sp. HOB1 was found to be highly potent in degrading higher concentrations of naphthalene under laboratory microcosms (Pathak et al., 2009). The growth of the isolated strains MAM-26 on different concentration of Naph. revealed that the presence of Naph. in BSM was toxic to the organism and the only growth increase had been recorded at 1000 and 2000 mg/L of Naph. after an incubation period of 14 days. At the first days of incubation, growth was decreased due to toxicity of Naph. as indicted in Table (14) and Figure (31). The results also revealed that the higher growth was found in case of higher concentrations after 14 days incubation. This may be explained on the bases that this prolonged period was for adaptation to overcome the substrate toxicity.

121 Results and Discussion

Table (14): Growth and extracellular protein of strain MAM- 26 on different concentrations of naphthalene.

Conc.

500(mg/l) 750(mg/l) 1000(mg/l) 1500(mg/l) 2000(mg/l) Incubation period(days) Protein Protein Protein Protein Protein OD OD OD OD OD Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.130 122.3 0.109 123.8 0.145 120.0 0.136 118.3 0.132 123.7 1 0.119 125.8 0.106 148.8 0.125 115.4 0.113 120.7 0.133 138.5 2 0.105 107.6 0.104 94.6 0.109 102.3 0.094 220.0 0.119 230.0 3 0.100 81.2 0.090 83.0 0.096 84.0 0.073 72.3 0.076 83.0 4 0.093 111.5 0.078 82.7 0.105 100.0 0.078 103.0 0.085 104.0 5 0.099 76.9 0.088 84.2 0.100 81.5 0.071 93.8 0.090 83.0 6 0.117 86.2 0.088 73.0 0.113 83.0 0.118 91.9 0.111 76.9 7 0.110 86.2 0.096 73.0 0.107 84.0 0.087 89.0 0.143 80.0 14 0.099 86.4 0.085 85.0 0.155 85.0 0.120 88.0 0.194 85.0 21 0.083 85.8 0.071 78.5 0.096 88.5 0.080 85.0 0.129 93.5

122 Results and Discussion

500(mg/l) 750(mg/l) 1000(mg/l) 0.25 1500(mg/l) 2000(mg/l)

0.2 ) 0.15 nm 600 . ( D

. 0.1 O

0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (31): Growth of strain MAM-26 on different concentrations of naphthalene.

250 500(mg/l) 750(mg/l) 1000(mg/l) 1500(mg/l) 2000(mg/l) 200

150

100 Protein (ug/ml)Protein

50

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (32): Extracellular protein of strain MAM-26 on different concentrations of naphthalene.

123 Results and Discussion

The results of extracellular protein secreted by the isolated strain MAM-26 on the higher concentrations of Naph. (1500 and 2000 mg/L) have been recorded after 2 days incubation period as shown in Figure (32).

Growth and extracellular protein formed by isolated strain MAM-43 on different concentrations of Naph. was indicated in Table (15) and Figure (33 and 34). From the results at the higher concentrations (1500 and 2000 mg/L) it is obvious that the first period of incubation till the sixth and seventh days the growth decreased. The only slightly increase have been recorded in case of higher concentrations (1500, 2000 mg/L) at the seventh and six days respectively.

The extracelular protein in case of 1500 mg/L Naph. reached its maximum production after 2 days, at 750 mg/L the maximum protein secretion was found after one day, while the increase in secretion of protein by MAM-43 on 2000 mg/L Naph. was recorded for the first and second days.

Growth of isolated strain MAM-62 on different concentrations of Naph. had been indicated by Table (16) and Figure (35). In this case, the bacterial strain growth was decreased at the first period of incubation. The increase in growth have been recorded at the seventh day at concentration 750, 1000 and 1500 mg/L Naph., while at 2000 mg/L the increase began at the sixth day.

124 Results and Discussion

Table (15): Growth and extracellular protein of strain MAM- 43 on different concentrations of naphthalene.

Conc.

500(mg/l) 750(mg/l) 1000(mg/l) 1500(mg/l) 2000(mg/l) Incubation period(days) Protein Protein Protein Protein Protein OD OD OD OD OD Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.167 111.5 0.172 110.8 0.184 112.0 0.166 108.0 0.170 114.0 1 0.125 125.4 0.144 159.2 0.124 129.2 0.125 128.8 0.140 134.6 2 0.118 113.8 0.139 120.0 0.102 109.2 0.120 219.2 0.130 136.0 3 0.107 85.4 0.141 90.0 0.095 93.8 0.129 110.0 0.119 84.6 4 0.107 89.2 0.145 97.6 0.110 107.7 0.134 91.1 0.103 105.7 5 0.123 86.9 0.155 103.8 0.120 84.6 0.129 87.7 0.128 91.5 6 0.132 76.2 0.159 95.4 0.137 88.5 0.149 90.7 0.174 77.3 7 0.152 78.0 0.169 96.0 0.158 87.0 0.173 96.0 0.159 84.0 14 0.121 83.0 0.149 97.0 0.161 86.0 0.142 97.0 0.175 86.0 21 0.117 88.5 0.173 99.6 0.131 85.4 0.163 100.4 0.173 96.2

125 Results and Discussion

500(mg/l) 750(mg/l) 1000(mg/l) 1500(mg/l) 2000(mg/l)

0.2 0.18 0.16 0.14 ) 0.12 nm 0.1 600 . ( .

D 0.08 . O 0.06 0.04 0.02 0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (33): Growth of strain MAM- 43 on different concentrations of naphthalene.

250 500(mg/l) 750(mg/l) 1000(mg/l) 1500(mg/l) 2000(mg/l)

200

150

100 Protein (ug/ml)

50

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (34): Extracellular protein of strain MAM- 43 on different concentrations of naphthalene.

126 Results and Discussion

The protein secretd by isolated strain MAM-62 revelaed that the higher concentrations (1500 and 2000 mg/L) reached their maximum after 2 days while the less concentrations (500, 750 and 1000) reached maximum production after 1 days as shown in Fgure (36).

The growth behavior of the isolated strain MAM-68 on different concentrations of Naph. showed that the only increase was found on cocnnetrations 750 and 1000 mg/L Naph. as indicated in Table (17) and Figure (37). The highest production of the extracellular protein was recorded after 2 days incubation at the higher concentrations (1500 and 2000 mg/L) as shown in Figure (38).

Growth pattern of isolated strain MAM-78 cleared that the only increase in all the concentrations at all the incubation periods was found at the second day on the lowest concentration 500 mg/L Naph. as indicated in Table (18) and Figure (39).

127 Results and Discussion

Table (16): Growth and extracellular protein of strain MAM- 62on different concentrations of naphthalene. Conc.

500(mg/l) 750(mg/l) 1000(mg/l) 1500(mg/l) 2000(mg/l) Incubation period(days) Protein Protein Protein Protein Protein OD OD OD OD OD Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.137 119.2 0.143 120.0 0.139 122.0 0.169 117.9 0.132 116.9 1 0.108 130.8 0.119 141.5 0.104 167.7 0.134 156.9 0.129 132.3 2 0.109 97.7 0.134 97.3 0.071 108.0 0.133 233.5 0.094 219.2 3 0.103 83.8 0.158 79.2 0.094 83.0 0.155 103.8 0.109 82.3 4 0.091 80.7 0.140 87.7 0.094 103.0 0.146 116.5 0.132 92.3 5 0.110 76.9 0.136 80.0 0.099 86.9 0.154 100.0 0.124 80.8 6 0.114 69.2 0.152 67.7 0.129 83.8 0.169 95.4 0.151 75.4 7 0.123 67.0 0.173 72.0 0.155 83.9 0.202 100.0 0.146 80.0 14 0.080 68.0 0.126 77.0 0.158 84.0 0.127 110.0 0.122 85.0 21 0.116 66.9 0.126 80.7 0.114 84.2 0.145 120.4 0.132 90.8

128 Results and Discussion

500(mg/l) 750(mg/l) 1000(mg/l) 0.25 1500(mg/l) 2000(mg/l)

0.2 ) 0.15 nm 600 . (

D 0.1 . O

0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (35): Growth of strain MAM- 62 on different concentrations of naphthalene.

250 500(mg/l) 750(mg/l) 1000(mg/l) 1500(mg/l) 2000(mg/l)

200

150

100 Protein (ug/ml)Protein

50

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (36): Extracellular protein of strain MAM-62 on different concentrations of naphthalene.

129 Results and Discussion

Table (17): Growth and extracellular protein of strain MAM-68 on different concentrations of naphthalene.

Conc.

500(mg/l) 750(mg/l) 1000(mg/l) 1500(mg/l) 2000(mg/l) Incubation period(days) Protein Protein Protein Protein Protein OD OD OD OD OD Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.165 132.3 0.144 130.0 0.171 129.0 0.158 131.0 0.151 132.0 1 0.122 134.6 0.111 126.1 0.140 134.6 0.113 143.5 0.130 137.7 2 0.137 108.0 0.111 104.6 0.126 117.7 0.104 237.6 0.103 218.4 3 0.121 83.8 0.127 83.8 0.135 76.9 0.079 92.7 0.103 76.9 4 0.122 79.9 0.118 95.4 0.140 98.5 0.084 113.4 0.099 103.8 5 0.137 72.3 0.128 79.6 0.147 71.5 0.087 101.0 0.108 73.0 6 0.136 67.7 0.142 68.8 0.173 78.8 0.116 87.3 0.134 65.4 7 0.132 70.0 0.164 68.0 0.195 79.0 0.122 88.0 0.142 70.0 14 0.127 70.0 0.142 67.9 0.113 80.0 0.159 95.0 0.112 80.0 21 0.124 72.3 0.131 66.9 0.165 81.2 0.121 96.2 0.134 83.0

130 Results and Discussion

500(mg/l) 750(mg/l) 1000(mg/l) 0.25 1500(mg/l) 2000(mg/l)

0.2 ) 0.15 nm 600 . ( D

. 0.1 O

0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (37): Growth of strain MAM- 68 on different concentrations of naphthalene.

250 500(mg/l) 750(mg/l) 1000(mg/l) 1500(mg/l) 2000(mg/l)

200

150

100 Protein (ug/ml)Protein

50

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (38): Extracellular protein of strain MAM-68 on different concentrations of naphthalene.

131 Results and Discussion

The results of protein secretion by MAM-78 revealed that the highest protein was found after 2 days incubation for the higher concentrations (1500 and 2000 mg/L) Naph. as indicated in Table (18) and Fig. (40).

The counts of the five selected isolated strains at the beginning (initial count) at zero time and that of the isolated strain in BSM amended by the five different concentrations of Naph. at the end of incubation period (21 days) was indicated in Table (19). The results revealed that non of the five concentrations in which the isolated strain MAM-26 had been grown more than that of the intial (4.4 x 107 CFU/ml) after 21 days incubation .

On contrast, the isolated strain MAM-43, its growth (count) on all the five Naph. concentrations was more than that of initial 1.0x105 CFU/ml. The highest count (2.0 x106 CFU/ml) were recorded at 2000 mg/L. Isolated strain MAM-62, non of its count on different concentration can exceed the initial (1.0 x106 CFU/ml). The only concentration (1000mg/L) of isolate MAM 68 that exceed the initial count was found to be 2.6x106 CFU /ml. However, isolate MAM-78 which cannot indicate obvious growth by O.D. revealed a greater count at concentrations 750, 1000 and 2000 mg/L Naph. The highest count (1.7x 106 CFU/ml) was recorded at 2000 mg/L.

This may be explained it’s activity secreting extracellular protein. This phenomena also explain why we use more than one parameter to determine the ability of certain strain to grow especially on toxic substrate like Naph. and degrade it.

132 Results and Discussion

Table (18): Growth and extracellular protein of strain MAM-78 on different concentrations of naphthalene.

Conc.

500(mg/l) 750(mg/l) 1000(mg/l) 1500(mg/l) 2000(mg/l) Incubation

period(days)

Protein Protein Protein Protein Protein OD OD OD OD OD Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.171 119.2 0.180 120.0 0.174 122.0 0.159 119.0 0.172 117.5 1 0.165 126.9 0.169 125.4 0.166 120.3 0.143 123.0 0.150 136.9 2 0.181 91.9 0.162 109.2 0.156 111.9 0.127 212.3 0.137 213.8 3 0.162 90.0 0.144 73.0 0.138 80.0 0.117 73.0 0.136 80.4 4 0.143 78.8 0.138 100.0 0.125 102.3 0.126 93.8 0.121 109.2 5 0.148 85.4 0.143 81.5 0.130 94.2 0.113 78.8 0.109 81.5 6 0.155 76.9 0.118 73.8 0.147 82.3 0.134 74.6 0.132 70.0 7 0.171 78.0 0.168 76.0 0.138 85.0 0.142 77.0 0.138 80.0 14 0.134 80.0 0.143 80.0 0.123 88.0 0.131 88.0 0.138 85.0 21 0.136 84.6 0.132 85.8 0.115 90.8 0.119 93.5 0.108 96.2

133 Results and Discussion

500(mg/l) 750(mg/l) 1000(mg/l)

0.2 1500(mg/l) 2000(mg/l) 0.18 0.16 0.14 ) 0.12 nm

600 0.1 . ( D

. 0.08 O 0.06 0.04 0.02 0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (39): Growth of strain MAM-78 on different concentrations of naphthalene.

250 500(mg/l) 750(mg/l) 1000(mg/l)

1500(mg/l) 2000(mg/l) 200

150

100 Protein Protein (ug/ml)

50

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (40): Extracellular protein of strain MAM-78 on different concentrations of naphthalene.

134 Results and Discussion

Table (19): Count of the selected isolated strains on different concentrations of naphthalene after 21 days incubation period.

Concentrations (mg/L) Isolate Initial count code 500 750 1000 1500 2000 (CFU/ml) Io

count ▲ logN count ▲ logN count ▲ logN count ▲ logN count ▲ logN count ▲ logN MAM- 44.0*106 7.64 25.0*105 6.39 8.0*105 5.90 16.5*105 6.21 1.0*104 4.00 1.0*104 4.00 26 MAM- 1.0*105 5.00 9.0*105 5.95 10.0*105 6.00 8.5*105 5.92 5.0*105 5.69 20.0*105 6.30 43 MAM- 10.0*105 6.00 3.0*105 5.47 1.5*105 5.17 2.5*105 5.39 2.0*105 5.30 2.0*105 5.30 62 MAM- 20.0*105 6.30 16.0*105 6.20 15.0*105 6.17 26.5*105 6.42 9.0*105 5.95 12.0*105 6.07 68 MAM- 7.0*105 5.80 4.5*105 5.65 9.0*105 5.95 9.0*105 5.95 5.0*105 5.69 17.0*105 6.23 78 ▲ count = CFU/ml

135 Results and Discussion

Plant germination and growth are strongly inhibited by the presence of volatile, water-soluble low molecular weight hydrocarbons (< 3 rings) such as benzene, toluene, xylene (BTX), , indene, nalphthalene and other possibly toxic substances. On the other hand, high molecular weight PAH (3- 5 rings) did not show any phytotoxicity under the conditions studied (Henner et al., 1999).

Naphthalaene was utilized for all the selected strains (B. Pumilus, B. Subtilis, M. Luteus, A. faecalis and Enterobacter sp.), whereas fluoranthene was only utilized by one strain affiliated to A. faecalis. Pyrene removing bacteria were only found in the genus Bacillus, a microbial group that also showed an increased phenanthrene-removal capacity (Toledo et al., 2006).

The ability of isolated strain MAM-62 to degrade Naph. in BSM during 21 days incubation period as indicated in Table (20) revealed that strain MAM-62 was the best naphthalene degrader. It degraded 97% of the highest concentration (2000 mg/L), although, its count in all concentrations, was lower than initial. On the other hand, isolated strain MAM-43, the count and the degradation were coincide. The results of degradation as indicated by Table (20) and Figure (41) proved that MAM-43 was the second best naphthalene degrader after MAM-62. It could degrade 95% of 2000 mg/L. Naph. in contrast of MAM-62 and MAM-43, the isolated strain MAM-78 was proved to be the worest one at concentration 2000 mg/L, inspit of its higher count (1.7 X106CFU/ml). The above results of Naph. In this study, especially their toxicity had been confirmed by the results of other investigators.

Anthracophyllum discolor was able to remove phenanthrene, anthracene, fluoranthene and pyrene in Kirk medium individually and in mixtures. The removal efficiency of anthracene (11.3%) and pyrene (17.5%)

136 Results and Discussion in the PAH mixture in liquid medium after 28 days was higher than that of the individual compounds (7.0% and 8.5% respectively), suggesting synergistic effects between PAHs or possible co-metabolism (Bauer and Capone, 1998 and Bouchez et al., 1995).

On the contrary, PAHs with low molecular weight such as 2-ring naphthalene and 3-ring phenanthrene were more susceptible to bacterial degradation than PAHs with more than 3- rings (Yu et al., 2005a).

PAHs were lost from all treatments with 38 C which being the optimum temperature for both PAH removal and microbial activity (Antizar- Ladislao, 2005). Toledo et al. (2006) characterized different bacterial strains, which were previously isolated from solid waste oil, with the capacity to growth on culture media supplemented with PAHs. These selected strains included a diversity of Gram-negative and Gram-positive bacteria with the capacity to grow on solid and liquid media amended with naphthalene, phenanthrene, fluoranthene or pyrene as carbon and energy source.

Growth of some strains in mineral liquid media amended with naphthalene, phenanthrene, fluoranthene or pyrene as sole carbon source was studied and our results showed that these strains can tolerate and remove different polycyclic aromatic hydrocarbons that may be toxic in the environment polluted with hydrocarbons (Toledo et al., 2006).

PAHs were shown to inhibit cell division of the two algae Nitzschia sp. and Skeletonema costatum. However, the basic activates of the two algae still remained (Hong et al.,2008).

It was found that the higher the PAH concentration, the lower the biomass (Ting et al., 2011).

137 Results and Discussion

Four series of batch experiments were conducted to investigate the effect of temperature, pH, naphthalene concentration and nitrate concentration on the naphthalene degradation under specific degradation condition. Our results showed that the degradation of naphthalene was most favorable at pH 7 and 25C (Lu et al., 2011). 3.5. Growth and degradation of phenanthrene by the most potent isolated strains

Growth of isolated strain MAM-26 on different concentration of Phen. was indicated in Table (21) and Figure (42). The growth showed an increase after six and seven days of incubation at 250 and 500 mg/L Phen. As the concentration increased more (750 mg/L), increase in growth began from the second day and continue to the end of incubation period. More increase in concentrations (1000 and 1500 mg/L) Phen., the growth began to increase at the sixth day and continue to the rest of incubation period, with maximum increase after 14 days at 1000 mg/L Phen., and after 7 days for 1500 mg/L Phen.

138 Results and Discussion

Table (20): Degradation percentage of naphthalene after 21 days by HPLC.

Degradation % Isolate code 500 750 1000 1500 2000 (mg/l) (mg/l) (mg/l) ( mg/l) (mg/l) MAM-26 95% 84% 15% 44% 49%

MAM-43 92% 45% 54% 32% 95%

MAM-62 78% 83% 88% 35% 97%

MAM-68 88% 9% 42% 49% 75%

MAM-78 68% 86% 55% 81% 7%

MAM-26 MAM-43 MAM-62 MAM-68 MAM-78

120%

100%

80%

60%

Degradation % 40%

20%

0% 500 750 1000 1500 2000 Concentration (mg/L)

Figure (41): Degradation percentage of naphthalene after 21 days by HPLC.

139 Results and Discussion

Table (21): Growth and extracellular protein of strain MAM-26 on different concentrations of phenanthrene. Conc.

250 (mg/l) 500 (mg/l) 750 (mg/l) 1000 (mg/l) 1500 (mg/l) Incubation period(days) Protein Protein Protein Protein Protein OD OD OD OD OD Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.178 116.9 0.133 117.8 0.145 118.0 0.213 116.9 0.150 115.0 1 0.182 96.2 0.133 130.7 0.170 133.8 0.121 186.9 0.142 140.7 2 0.148 215.4 0.108 214.6 0.190 216.9 0.130 219.2 0.077 215.3 3 0.147 99.2 0.095 90.7 0.181 75.4 0.140 83.8 0.116 84.6 4 0.149 76.2 0.086 81.5 0.189 96.2 0.161 96.9 0.112 96.2 5 0.143 80.7 0.073 80.8 0.191 104.6 0.139 82.3 0.109 76.5 6 0.200 67.7 0.159 73.0 0.278 73.8 0.265 75.3 0.300 67.7 7 0.199 60.0 0.173 74.0 0.345 77.0 0.215 77.0 0.710 70.0 14 0.173 55.0 0.140 75.0 0.285 84.0 0.310 79.0 0.650 77.0 21 0.149 44.6 0.172 79.2 0.315 88.5 0.296 81.5 0.505 86.2

140 Results and Discussion

Figure (42): Growth of strain MAM- 26 on different concentrations of phenanthrene.

Figure (43): Extracellular protein of strain MAM-26 on different concentrations of phenanthrene.

141 Results and Discussion

All the five concentrations of Phen. induced the maximum extracellular protein production after 2 days incubation as shown in Figure (43).

The growth profile of the isolated strain MAM-43 as indicated in Table (22) and Figure (44) revealed that, first concentration of Phen. (250 mg/L) have not shown any increase along all the incubation period but it was decreased. The other four concentrations showed an increase in growth from the seven days till the end of the incubation period. Concentrations (500, 750 and 1000 mg/L) Phen. showed their maximum growth at the seventh day, while at higher concentration (1500 mg/L) the highest growth was delayed to be after 21 days incubation period.

The extracellular protein of MAM-43 gave the same behavior of MAM-26, all the five concentrations produced the maximum secretion of protein at the second day as shown in Figure (45).

Trend of growth of the isolated strain MAM-62 as indicated from Table (23) and Figure (46) revealed that, the increase in all the five concentrations began from the sixth day.

142 Results and Discussion

Table (22): Growth and extracellular protein of strain MAM-43 on different concentrations of phenantherene

Conc.

250 (mg/l) 500 (mg/l) 750 (mg/l) 1000 (mg/l) 1500 (mg/l) Incubation period(days) Protein Protein Protein Protein Protein OD OD OD OD OD Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.162 105.4 0.161 110.0 0.174 102.0 0.252 105.0 0.172 107.8 1 0.160 100.7 0.132 126.9 0.142 115.4 0.173 134.6 0.138 136.9 2 0.103 215.0 0.117 210.0 0.095 224.6 0.174 223.0 0.127 215.3 3 0.116 90.0 0.102 86.9 0.152 83.8 0.194 88.4 0.130 81.2 4 0.089 66.2 0.094 83.0 0.156 91.5 0.194 92.3 0.138 90.7 5 0.094 76.4 0.101 79.6 0.106 90.7 0.238 96.2 0.125 85.4 6 0.132 46.9 0.130 69.2 0.172 85.4 0.245 66.2 0.289 69.2 7 0.145 49.0 0.195 69.0 0.285 86.0 0.450 66.0 0.405 74.0 14 0.122 56.0 0.175 67.0 0.205 86.0 0.390 80.0 0.397 80.0 21 0.114 62.6 0.176 67.7 0.191 86.9 0.355 89.2 0.435 86.5

143 Results and Discussion

0.5 250 (mg/l) 500 (mg/l) 750 (mg/l) 1000 (mg/l) 0.45 1500 (mg/l) 0.4

0.35 0.3 nm)

600 600 0.25 0.2 O.D. ( O.D. 0.15 0.1

0.05 0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (44): Growth of strain MAM-43 on different concentrations of phenanthrene.

Figure (45): Extracellular protein of strain MAM-43 on different concentrations of phenanthrene.

144 Results and Discussion

The lowest two concentrations (250 and 500 mg/L) Phen. reached their maximum growth at the seventh day. More increase in concentration (750 mg/L) Phen. needed more time to reach maximum growth (14 days). More and more increase in concentrations (1000 and 1500 mg/L) Phen. was more delay i.e. 21 days of incubation to reach maximum growth.

Maximum extracellular protein of MAM-62 had been recorded at the second day as the same trend in case of isolates MAM-26 and MAM-43 for all the five concentrations as shown in Figure (47).

The growth trend of isolated strain MAM-68 on different concentrations of Phen. as shown in Table (24) and Figure (48) indicated that growth on all concentration began at the sixth day and reached the maximum growth at the seventh day. The extracellular protein secretion by isolate MAM-68 reached the maximum productivity at the second day for all concentration as shown in Figure (49).

The behavior of growth of the isolated strain MAM-78 as shown in Table (25) and Figure (50) revealed that growth was began at the sixth day for the three higher concentrations of Phen. (750, 1000 and 1500 mg/L), but the highest growth for all the four concentrations have been recorded at the seventh day except for concentration ratio 1000 mg/L Phen; where the maximum had been reached at the day 14.

145 Results and Discussion

Table (23): Growth and extracellular protein of strain MAM-62 on different concentrations of phenanthrene. Conc.

250 (mg/l) 500 (mg/l) 750 (mg/l) 1000 (mg/l) 1500 (mg/l) Incubation period(days) Protein Protein Protein Protein Protein Zero OD OD OD OD OD (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) time 0.195 123.0 0.138 120.0 0.192 118.0 0.161 123.0 0.129 125.0 1 0.195 144.6 0.132 131.2 0.155 142.7 0.166 138.5 0.103 138.8 2 0.145 230.8 0.104 215.4 0.118 218.5 0.160 219.2 0.101 226.1 3 0.170 100.7 0.102 83.8 0.126 73.0 0.157 84.6 0.141 65.4 4 0.161 83.0 0.111 82.3 0.128 126.9 0.190 88.5 0.139 94.6 5 0.164 86.9 0.106 75.4 0.131 95.4 0.143 91.5 0.129 81.5 6 0.220 66.0 0.148 64.6 0.230 80.8 0.292 69.2 0.270 61.9 7 0.245 76.0 0.199 70.0 0.215 85.0 0.405 73.0 0.340 65.0 14 0.215 80.0 0.180 77.0 0.235 87.0 0.360 77.0 0.280 77.0 21 0.165 81.5 0.185 84.6 0.218 91.2 0.425 87.7 0.410 86.9

146 Results and Discussion

0.6 250 mg/l 500 mg/l 750 mg/l 1000 mg/l 1500 mg/l 0.5 ) 0.4 nm 600

.( 0.3 D . O 0.2

0.1

0 0 1 2 3 4 5 6 7 14 21

Incubation time (days)

Figure (46): Growth of strain MAM-62 on different concentrations of phenanthrene.

250 250 mg/l 500 mg/l 750 mg/l 1000 mg/l 1500 mg/l 200

150

100 Protein (ug/ml)

50

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (47): Extracellular protein of strain MAM- 62 on different concentrations of phenanthrene.

147 Results and Discussion

Table (24): Growth and extracellular protein of strain MAM-68 on different concentrations of phenanthrene. Conc.

250 (mg/l) 500 (mg/l) 750 (mg/l) 1000 (mg/l) 1500 (mg/l) Incubation period(days) Protein Protein Protein Protein Protein OD OD OD OD OD Zero (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) time 0.169 114.6 0.140 112.9 0.164 110.8 0.293 114.0 0.169 116.0 1 0.161 116.9 0.147 101.5 0.135 134.3 0.160 138.5 0.131 138.3 2 0.166 189.2 0.119 206.2 0.190 220.0 0.155 226.2 0.134 228.5 3 0.157 79.2 0.137 79.2 0.133 76.9 0.148 80.0 0.138 63.8 4 0.156 68.0 0.161 79.6 0.157 104.6 0.183 100.0 0.142 90.4 5 0.146 65.4 0.113 75.4 0.151 89.2 0.184 102.3 0.156 88.5 6 0.180 49.2 0.176 57.7 0.241 76.9 0.258 73.0 0.310 69.2 7 0.203 55.0 0.192 63.0 0.300 80.0 0.355 73.0 0.475 77.0 14 0.163 63.0 0.180 70.0 0.285 84.0 0.300 80.0 0.365 83.0 21 0.152 68.1 0.212 80.7 0.300 88.0 0.285 85.0 0.475 89.2

148 Results and Discussion

0.6 250 mg/l 500 mg/l 750 mg/l 1000 mg/l 0.5 1500 mg/l

0.4

) 0.3 nm 600

. ( 0.2 D . O 0.1

0 0 1 2 3 4 5 6 7 14 21

Incubation period (days)

Figure (48): Growth of strain MAM- 68 on different concentrations of phenanthrene.

250 250 mg/l 500 mg/l 750 mg/l 1000 mg/l 200 1500 mg/l

150

100 Protein (ug/ml)Protein 50

0 0 1 2 3 4 5 6 7 14 21

Incubation period (days)

Figure (49): Extracellular protein of strain MAM-68 on different concentrations of phenanthrene.

149 Results and Discussion

As usual in all the above mentioned results, the same trend for all the isolates, the maximum extraclellular protein had been recorded at the second day for all the used concentrations as shown in Figure (51).

The count of the five isolated strains at the end of the incubation period as indicated in Table (26) revealed that non of the five concentrations of Phen. caused an increase in count more than the initial count (4.4 X107 CFU/ml) for the isolated strain MAM-26. However, isolate MAM-43 showed an increase in count (1.3x106, 1.4x106 and 2.0x105 CFU/ml) for concentrations 250, 1000 and 1500 mg/L Phen. respectively.

Non of the five concentrations of Phen. increased the counts of both isolates MAM-62 and MAM-68, but strain MAM-78, there were an increase in counts of concentrations 250, 500 and 100mg/L phen. Although, non of the five contrations of Phen. their counts exceed the count of initial (1.0x106CFU/ml) in case of isolated strain MAM-62 this isolate MAM-62 was the best phenanthrene degrading strain as indicated in Table (27) and Figure (52). Strain MAM-62 degraded 89% of 250 mg/L Phen. It can also degraded 96% and 98% of 1000 and 1500 mg/L phen respectively. The second best degrader of phenantherene was isolate MAM43. The worest one in Phen. degradation was isolate MAM-78.

The previous results in this study indicated that each bacterial isolated strain having its own behavior in degrading phenanthrene via secreting a battery of extracellular enzymes as expressed by extracellular protein which was determined in this study. The activities of these enzymes differ from strain to another. The degradation percentage depends mainly on the enzymatic activity not on count of cells. So the best phenanthrene degrader was MAM-62 followed by MAM-43.

All the above results can be confirmed and explained by the researches of other investigators as the following.

150 Results and Discussion

Table (25): Growth and extracellular protein of strain MAM-78 on different concentrations of phenanthrene.

Conc.

250 (mg/l) 500 (mg/l) 750 (mg/l) 1000 (mg/l) 1500 (mg/l) Incubation period(days) Protein Protein Protein Protein Protein OD OD OD OD OD Zero (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) time 0.191 165.4 0.181 166.9 0.179 170.0 0.204 165.0 0.212 174.0 1 0.182 115.4 0.189 107.7 0.175 118.0 0.186 153.8 0.142 151.2 2 0.163 207.7 0.153 207.7 0.137 145.4 0.237 230.7 0.128 220.4 3 0.167 86.2 0.140 68.5 0.137 73.0 0.190 92.3 0.122 94.2 4 0.166 76.9 0.131 68.0 0.142 120.0 0.242 127.7 0.165 84.6 5 0.157 69.2 0.137 76.9 0.128 92.3 0.194 99.6 0.157 100.7 6 0.180 62.0 0.160 63.0 0.222 69.2 0.260 95.8 0.300 74.6 7 0.202 66.0 0.240 64.0 0.360 73.0 0.285 100.0 0.415 77.0 14 0.163 70.0 0.192 70.0 0.212 80.0 0.325 103.0 0.322 80.0 21 0.119 79.2 0.169 77.7 0.192 83.8 0.305 106.2 0.357 86.9

151 Results and Discussion

250 (mg/l) 500 (mg/l) 0.45 750 (mg/l) 1000 (mg/l) 1500 (mg/l) 0.4

0.35

) 0.3

nm 0.25 600

. ( 0.2 D .

O 0.15

0.1

0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (50): Growth of strain MAM- 78 on different concentrations of phenanthrene.

250 250 (mg/l) 500 (mg/l) 750 (mg/l) 1000 (mg/l) 1500 (mg/l)

200

150

100 Protein (ug/ml)Protein

50

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (51): Extracellular protein of strain MAM-78 on different concentrations of phenanthrene.

152 Results and Discussion

Table (26): Count of the selected isolated strain on different concentrations of phenanthrene after 21 days incubation period.

Concentrations (mg/L) Isolate Zero time 250 500 750 1000 1500 code initial (Io) count▲ logN count▲ logN count▲ logN count▲ logN count▲ logN count▲ logN MAM- 4.4*107 7.64 9.6*106 6.98 5.5*106 6.74 1.5*107 7.17 1.65*107 7.21 1.0*104 4.00 26 MAM- 1.0*105 5.00 1.3*106 6.11 0.3*105 4.47 1.0*105 5.0 1.4*106 6.146 2.0*105 5.30 43 MAM- 1.0*106 6.00 2.5*105 5.39 3.0*105 5.47 4.5*105 5.65 2.5*105 5.39 2.9*105 5.46 62 MAM- 2.0*106 6.30 1.0*106 6.00 7.5*105 5.87 9.5*105 5.97 8.5*105 5.92 1.2*106 6.07 68 MAM- 7.0*105 5.80 3.0*106 6.47 1.35*106 6.13 3.5*105 5.54 8.5*105 5.92 5.0*105 5.69 78

▲count = CFU/ml

153 Results and Discussion

Huesemann et al. (2004) found that phenanthrene, although readily bioavailable, was not immediately biodegraded because a lag period of approximately 7 days was required to build up sufficiently high numbers of phenathrene degrading bacteria.

Yuan et al. (2000) observed that the biodegradation of polycyclic aromatic hydrocarbons (PAHs) by an aerobic mixed culture utilizing phenanthrene as its carbon source. The optimal conditions determined as 30C and pH 7.0.

Viamajala et al. (2007) found that three thermophilic Geobacilli were isolated from that grew on phenathrene at 60C and degraded the PAH more rapidly than other reported mesophiles.

After the initial lag, phenanthrene degradation was rapid, such that, 5 mg1-1 phenanthrene was removed from solution within 4-6 h. after one set of degradation (Viamajala et al., 2007).

A series of phenanthrene-degrdation tests were carried out at various pH from 6.0 to 8.0 and temperatures from 20 to 37C. the optimal conditions for phenanthrene degradation was found to be at pH 7.0 and 30 C. the results indicated that the degradation rate of strain ZP1 decreased while phenanthrene ranged from 250 to 1000 ppm (Zhao et al., 2008).

Aeration provided the most rapid treatment and resulted in almost complete removal of phenathrene after 28 days (Muckian et al., 2009).

All isolates had a similar optimal growth temperature (25C) and optimal growth pH (7.0) in a minimal salt medium (MSM) with 0.1% (W/V) phenanthrene as the sole source of carbon and energy (Change et al., 2011).

154 Results and Discussion

Almost all of the phenanthrene was consumed during the first 60 h. When the cells were grown on 20, 50, 100 and 200 mg/L of phenanthrene, the PDO activity increased with a decrease of phenanthrene concentration, showing a characteristic peak at a later stage of degradation, i.e. at 48, 48, 56, and 56h, respectively. And the characteristic peak of C23O activity appeared at 72, 72, 64 and 72h, respectively. These results suggest a delicate mechanisms in the regulation of phenantrhene-degrading enzymes in this strain (Tian et al., 2002).

Following an initial 3-5 h lag phase the mixed culture completely degraded the phananthrene in a 5 mg/l solution within 28 h. A plate count of cell numbers revealed a range from 2.2x106 to 8.4x108, indicating the strain’s ability to utilize phenanthrene as a carbon source (Yuan et al., 2000).

The low solubility of phenanthrene was limiting the growth (Andreoni et al., 2004).

The decreasing biodegradation rate per gram of phenanthrene beyond 1 g/L of phenanthrene concentration may be due to the increased level of toxic metabolite(s) generated during the degradation process (Mallick and Dutta, 2008).

All cultures degraded phenanthrene without the appearance of any metabolites in culture broths. The protein content patterns of culture broths confirmed the ability of strains to utilize phenanthrene as the sole C source (Andreoni et al., 2004).

The result showed that phenanthrene was the best substrate to support bacterial growth independently of the strain tested (Toledo et al., 2006).

The results of GC analyses show that strain ZP1 can nearly degraded all phenanthrene within 8 days for one substrate for each bacterium, the biomass increased as the remaining concentration of the substrate decreased (Zhao et al., 2008).

155 Results and Discussion

Table (27): Degradation percentage of phenanthrene after 21 days by HPLC.

Degradation % Isolate code 250 mg/l 500 mg/l 750 mg/l 1000 mg/l 1500 mg/l

MAM-26 20% 51% 66% 73% 50%

MAM-43 29% 37% 41% 60% 97%

MAM-62 89% 70% 81% 96% 98%

MAM-68 59% 40% 70% 54% 78%

MAM-78 32% 77% 25% 29% 7%

MAM-26 MAM-43 MAM-62 MAM-68 MAM-78

120%

100%

80%

60%

Degradation Degradation % 40%

20%

0% 250 500 750 1000 1500 Concentration (mg/L)

Figure (52): Degradation percentage of phenanthrene after 21 days by HPLC.

156 Results and Discussion

Individual phenanthrene and dibenzothiophene compound losses due to abiotic phenomena are less than 20% even after 18 days of experiment. In the degradation flasks, after 6 and 11 days of experiment, 86 and 62% respectively of the total amount of phenanthrene and dibenzothiopehen compounds initially introduced are still present. After 14 days, the remaining percentage of total phenanthrene and dibenzothiophene compounds was around 45% and remained constant until the end of the experiment (Mazeasa et al., 2002).

More than 90% of Phen. was degraded by strain GY2B, which could grow well and utilize Phen. as sole carbon source. The degradation of Phen. was favored in slightly basic media at 25- 30C at 100 mg/L (Tao et al., 2007).

The result also indicated that the degradation rate of phenenthrene ranged from 250 to 1000 ppm with strain ZP1 remained nearly the same, i.e., a high concentration of phenanthrene did not inhibit phenanthrene- degradation ability (Zhao et al., 2008).

Pseudomonas sp. CH-11 and Staphylococcus sp. KW-07 degraded 90% of added phenanthrene in 3 days and Ochrobactrum sp. CH-19 degraded 90% of the phenanthrene in 7 days under laboratory batch culture conditions. After inoculation of 1x1011 cells of Staphyloccous sp. KW-07, over 90% degradation of 0.1% phenanthrene (0.1 g/100 g soil) was achieved after 1 month at 25C (Chang et al., 2011).

The effect of initial PAH concentration on PAH degradation by G. lucidum was further evaluated. The presence of high concentrations (50 or 100 mg/1) of PAHs, both phenanthrene and pyrene decreased rapidly during the first 6 d of incubation. More than 95% of 50 mg /l and around 50% of 100 157 Results and Discussion mg /l PAHs disappeared during this period. The concentrations of PAHs did not decline significantly afterward. At low concentrations (2 or 20 mg/L), degradation of both PAHs were most active during the first 3 d of incubation. On the 6 th day, neither phenanthrene nor pyrene was detected in the cultures with 2 mg/L PAHs and the residual concentrations of both PAHs dropped to less than 5% in the cultures with 20mg/L PAHs (Ting et al., 2011).

3.6. Growth and degradation of anthracene by the most potent isolated strains. Growth of isolated strain MAM-26 on different concentrations of anthracene (Anth.) have been indicated in Table (28) and Figure (53). The results revealed that isolate MAM-26 reached maximum growth at second day of incubation at concentration 40 mg/L Anth. However, at higher concentration (50 mg/L) the highest growth recorded after one day of incubation, but till the sixth day, the growth was greater than the initial inoclum. With more increase of concentrations (75, 100 and 150 mg/L), the highest growth on Anth. was recorded at second, third and fifth days of incubation respectively.

Extracellular protein secreted by isolated strain MAM-26 grown on different concentrations of Anth. was indicated in Table (28) and Figure (54). It was clear that all concentrations increased the secretion of extracellular proteins at the first day and began to decrease to reach the lowest value at the second day, then began to increase gradually at the rest of the incubation period. But, the lowest concentration (40mg/L) Anth. began the decrease from the beginning and continue the decrease till the third day and then began again the increase to reach the maximum extracellular protein at the end of incubation period.

158 Results and Discussion

Table (28): Growth and extracellular protein of strain MAM- 26- on different concentrations of anthracene. Conc. 40 50 75 100 150 Incubation ( mg/l) (mg/l) (mg/l) (mg/l) (mg/l) Period(days) Protein Protein Protein Protein Protein OD OD OD OD OD Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.157 30.8 0.143 30.0 0.156 28.3 0.168 30.7 0.162 33.0 1 0.169 30.8 0.169 38.5 0.178 33.8 0.185 34.6 0.180 34.6 2 0.170 27.7 0.164 21.5 0.192 20.0 0.182 16.9 0.177 12.3 3 0.158 20.0 0.151 21.5 0.182 16.9 0.189 21.1 0.166 21.5 4 0.159 25.0 0.150 27.0 0.180 21.0 0.180 20.0 0.180 23.0 5 0.159 26.2 0.153 29.6 0.177 26.9 0.175 20.7 0.200 26.2 6 0.147 27.0 0.145 30.0 0.170 30.0 0.174 28.0 0.180 28.0 7 0.130 33.0 0.143 31.0 0.164 33.0 0.173 33.0 0.173 32.0 14 0.136 37.0 0.140 32.0 0.158 38.0 0.171 40.0 0.158 36.0 21 0.126 57.7 0.132 33.8 0.147 40.0 0.152 46.1 0.144 38.5

159 Results and Discussion

0.35 40 mg/l 50 mg/l 0.3 75 mg/l 100 mg/l 150 mg/l 0.25 ) 0.2 nm

600 0.15 . ( D .

O 0.1 0.05 0 0 1 2 3 4 5 6 7 14 21

Incubation period (days)

Figure (53): Growth of strain MAM-26 on different concentrations of anthracene.

70 40 mg/l 50 mg/l 75 mg/l 100 mg/l 60 150 mg/l

50

40

30

20 Protein ug/mlProtein 10

0 0 1 2 3 4 5 6 7 14 21

Incubation period (days)

Figure (54): Extracellular protein of strain MAM-26 on different concentrations of anthracene.

160 Results and Discussion

The trend of growth of the isolated strain MAM-29 on different concentrations of Anth. as indicated in Table (29) and Figure (55) revealed that, there was an increase in growth varied along the incubation periods. This increase depending on the concentrations of Anth; but the highest growth had been recorded at the first day of incubation for the five different concentrations, except the lowest concentration reached the highest growth at the fifth day. The behavior of extracellular protein secretion by isolate MAM- 29 was shown in Table (29) and Figure (56). This isolate showed the same trend of extracellular protein as shown for isolate MAM-26. There was an increase in protein secretion at the first day followed by a decrease at the second day and with more increase in incubation period, the protein began again to increase gradually to reach the maximum secretion at the end of incubation period.

Table (30) and Figure (57) revealed that growth of isolated strain MAM-43 on different concentrations of Anth. reached the maximum growth after the first day of incubation and began to decrease after that except at concentration 75 and 150 mg/L, there was no increase from the beginning but began to decrease.

However, the profile of the extracellular protein was found to follow the same trend of isolates MAM-26 and MAM-29 this means that, extracellular protein was increased at the first day of incubation and began to decrease till the second and third days, then began to increase gradually till it reached the maximum productivity at the end of incubation period as shown in Table (30) and Figure (58).

161 Results and Discussion

Table (29): Growth and extracellular protein of strain MAM-29 on different concentrations of anthracene. Conc.

40 50 75 100 150

(mg/l) (mg/l) (mg/l) (mg/l) (mg/l) Incubation period(days) Protein Protein Protein Protein Protein OD OD OD OD OD Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.112 41.5 0.120 43.0 0.116 46.0 0.140 41.5 0.122 39.6 1 0.127 43.8 0.132 49.2 0.151 48.5 0.162 45.8 0.158 47.7 2 0.125 40.0 0.126 30.8 0.149 22.3 0.152 23.0 0.158 23.1 3 0.121 30.7 0.122 27.7 0.133 20.7 0.149 46.2 0.144 33.0 4 0.123 34.0 0.118 30.0 0.120 27.0 0.150 40.0 0.144 33.0 5 0.134 38.5 0.116 36.9 0.130 32.3 0.153 33.0 0.145 36.9 6 0.110 44.0 0.106 38.0 0.125 45.0 0.148 38.0 0.140 40.0 7 0.100 60.0 0.099 39.0 0.122 60.0 0.139 44.0 0.138 44.0 14 0.099 70.0 0.096 42.0 0.123 70.0 0.137 48.0 0.123 50.0 21 0.093 84.2 0.097 45.0 0.115 96.0 0.141 50.0 0.110 56.1

162 Results and Discussion

40 mg/l 50 mg/l 75 mg/l 100 mg/l 150 mg/l 0.2

) 0.15 nm 600 .( D

. 0.1 O

0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (55): Growth of strain MAM-29 on different concentrations of anthracene.

120 40 mg/l 50 mg/l 75 mg/l

100 100 mg/l 150 mg/l

80

60

40 Protein (ug/ml)

20

0 0 1 2 3 4 5 6 7 14 21

Incubation period (days)

Figure (56): Extracellular protein of strain MAM-29 on different concentrations of anthracene.

163 Results and Discussion

Table (30): Growth and extracellular protein of strain MAM-43 on different concentrations of anthracene.

Conc. 40 50 75 100 150 Incubation (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) Period(days) Protein Protein Protein Protein Protein OD OD OD OD OD Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.211 45.0 0.219 44.9 0.237 43.5 0.234 44.6 0.231 42.0 1 0.228 51.5 0.234 53.0 0.237 51.9 0.256 47.7 0.217 56.9 2 0.205 47.3 0.182 42.3 0.192 44.6 0.185 33.8 0.181 35.4 3 0.170 34.6 0.164 26.1 0.141 29.2 0.175 35.4 0.152 38.5 4 0.165 36.0 0.162 30.9 0.160 30.0 0.183 36.0 0.150 40.0 5 0.161 38.5 0.160 38.5 0.163 42.3 0.188 36.2 0.146 46.2 6 0.158 40.0 0.157 44.0 0.155 50.0 0.180 40.0 0.153 50.0 7 0.144 50.0 0.154 50.0 0.150 66.0 0.175 42.0 0.160 53.0 14 0.139 60.0 0.153 52.0 0.144 70.0 0.171 44.0 0.168 58.0 21 0.136 67.3 0.148 55.4 0.135 83.0 0.177 49.9 0.131 61.5

164 Results and Discussion

0.3 40 mg/l 50 mg/l 75 mg/l 100 mg/l 150 mg/l 0.25 ) 0.2 600 (

0.15

0.1 Optical density Optical density

0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (57): Growth and extracellular protein of strain MAM-43 on different concentrations of anthracene.

90 40 mg/l 50 mg/l 75 mg/l 80 100 mg/l 150 mg/l 70 60 50 40 30

Protein (ug/ml) 20 10 0 0 1 2 3 4 5 6 7 14 21

Incubation period (days)

Figure (58): Extracellular protein of strain MAM-43 on different concentrations of anthracene.

165 Results and Discussion

Growth of isolated strain MAM-62 on different concentrations of Anth. was indicated in Table (31) and Figure (59). From the results, it was clear that growth was increased at the first day of incubation, then began to decrease at all the five concentrations. The trend of extracellular protein follow the same trend for the previous isolated strains, MAM-26, MAM-29, MAM-43, where the extracellular protein increased at the first day, then decreased at the second day and began to increased gradually till the end of the incubation period.

The difference in case of MAM-62 that in some concentrations their extracellular protein did not increased at the first day but began to decrease till the second day and then began to increase gradually as perivously mentioned and as indicated in Table (31) and Figure (60).

The growth trend of isolated strain MAM-68 differed completely in their growth as indicated in Table (32) and Figure (61). The results of this strain revealed that the maximum growth on the five concentrations of Anth. was found at the first day.

166 Results and Discussion

Table (31): Growth and extracellular protein of strain MAM-62 on different concentrations of anthracene. Conc.

40 50 75 100 150

(mg/l) (mg/l) (mg/l) (mg/l) (mg/l) Incubation Period(days) Protein Protein Protein Protein Protein OD OD OD OD OD Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.253 51.9 0.244 50.6 0.283 49.6 0.274 51.9 0.272 53.7 1 0.268 43.8 0.249 50.4 0.304 50.8 0.286 59.6 0.300 48.0 2 0.250 28.5 0.235 38.0 0.283 37.7 0.259 31.2 0.286 41.5 3 0.230 36.2 0.207 38.4 0.257 31.5 0.243 34.6 0.262 44.6 4 0.217 30.0 0.212 37.0 0.250 36.0 0.230 35.0 0.258 42.0 5 0.215 29.2 0.212 35.8 0.243 38.5 0.235 36.2 0.251 41.5 6 0.200 33.0 0.200 40.0 0.230 40.0 0.229 40.0 0.230 50.0 7 0.190 38.0 0.189 44.0 0.220 44.0 0.200 44.0 0.210 60.0 14 0.174 40.0 0.163 49.0 0.212 49.0 0.198 49.0 0.200 70.0 21 0.191 43.8 0.150 57.7 0.156 52.3 0.174 56.9 0.213 97.3

167 Results and Discussion

0.35 40 mg/l 50 mg/l 75 mg/l 0.3 100 mg/l 150 mg/l

0.25 ) 0.2 nm 600

.( 0.15 D . O 0.1

0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period(days)

Figure (59): Growth of strain MAM-62 on different concentrations of anthracene.

120 40 mg/l 50 mg/l 75 mg/l 100 mg/l 150 mg/l 100

80

60

40 Protein (ug/ml)

20

0 0 1 2 3 4 5 6 7 14 21

Incubation period (days)

Figure (60): Extracellular protein of strain MAM-62 on different concentrations of anthracene.

168 Results and Discussion

Table (32): Growth and extracellular protein of strain MAM-68 on different concentrations of anthracene. Conc.

40 50 75 100 150

(mg/l) (mg/l) (mg/l) (mg/l) (mg/l) Incubation period(days) Protein Protein Protein Protein Protein OD OD OD OD OD Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.164 46.2 0.155 44.8 0.182 42.0 0.174 46.2 0.176 48.0 1 0.177 34.6 0.185 37.3 0.209 42.7 0.205 44.6 0.230 46.2 2 0.175 19.2 0.169 25.4 0.205 25.8 0.190 25.4 0.205 29.2 3 0.168 33.0 0.160 28.8 0.176 23.0 0.184 30.8 0.207 36.9 4 0.155 33.0 0.165 27.0 0.173 25.0 0.185 28.0 0.190 33.0 5 0.147 29.2 0.169 27.7 0.171 30.8 0.187 25.4 0.176 33.8 6 0.140 33.0 0.160 28.0 0.166 33.0 0.180 30.0 0.170 38.0 7 0.135 37.0 0.150 33.0 0.163 38.7 0.175 34.0 0.165 43.0 14 0.133 40.0 0.144 39.0 0.161 44.8 0.169 40.0 0.164 50.0 21 0.148 40.0 0.139 46.2 0.120 50.8 0.149 42.3 0.146 53.8

169 Results and Discussion

40 (mg/l) 50 (mg/l) 75 (mg/l) 0.25 100 (mg/l) 150 (mg/l)

0.2 ) 0.15 nm 600

. ( 0.1 D . O

0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (61): Growth of strain MAM-68 on different concentrations of anthracene.

60 40 (mg/l) 50 (mg/l) 75 (mg/l)

100 (mg/l) 150 (mg/l) 50

40

30 Protein Protein (ug/ml) 20

10

0 0 1 2 3 4 5 6 7 14 21

Incubation period (days)

Figure (62): Extracellular protein of strain MAM-68 on different concentrations of anthracene.

170 Results and Discussion

The profile of secretion of extracellular protein revealed that there was decease in protein at the beginning till the second day following by slight increase till the rest of incubation period as indicated by Table (32) and Figure (62).

Another growth trend for the isolated strain MAM-78 was indicated in Table (33) and Figure (63). The maximum growth have been reached after 24 hours incubation and the growth began to decrease in case of growth on 40 and 100 mg/L Anth. However, the extracellular protein secreted by this strain decreased from the beginning till the 6th or 7th days of incubation and then began to increase again as shown in Table (33) and Figure (64).

These results followed the same trend for extracellular protein production by the different isolated strains when grown on different concentrations of anthracene as indicated in Figure (64).

The growth of the isolated strains Enterobacter Cloacae MAM-4 revealed that the highest growth on different concentrations of Anth. as indicated in Table (34) and Figure (65) was recorded at the second day of incubation except for the highest concentration (150 mg/L). The highest growth had been recorded at fifth day. However, the increase in growth had been recorded along the incubation period.

The extracellular protein secretion profile revealed that there was an increase in protein production at the first day followed by decrease at the second day followed by gradual increase in the following days till the end of the incubation period as indcated by Table (34) and Figure (66).

From all the above results, it is clear that every isolated strain have a special profile of growth, but the growth was not sufficient, it was increased at the first or second day and began to decrease. Also, the difference in growth (O.D) of each isolated strain from the initial inoculum (O.D) is not represent great difference.

171 Results and Discussion

Table (33): Growth and extracellular protein of strain MAM-78 on different concentrations of anthracene. Conc.

40 (mg/l) 50 (mg/l) 75 (mg/l) 100 (mg/l) 150 (mg/l) Incubation period(days) Zero time Protein Protein Protein Protein Protein OD OD OD OD OD (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.091 70.8 0.096 72.0 0.111 68.0 0.105 70.8 0.099 71.5 1 0.110 47.7 0.111 42.3 0.119 51.5 0.143 56.2 0.147 53.0 2 0.107 38.5 0.116 32.3 0.123 42.3 0.136 21.2 0.157 34.6 3 0.110 37.7 0.104 30.7 0.129 22.3 0.135 34.6 0.131 42.3 4 0.106 33.0 0.110 27.7 0.119 30.4 0.134 35.4 0.136 31.1 5 0.104 33.0 0.114 27.7 0.119 30.4 0.134 35.4 0.137 31.1 6 0.100 39.0 0.110 30.0 0.109 38.0 0.127 40.6 0.130 44.0 7 0.098 44.0 0.106 40.7 0.117 44.0 0.120 50.0 0.125 50.0 14 0.099 49.0 0.103 49.9 0.119 49.0 0.113 55.0 0.120 52.0 21 0.114 55.0 0.109 61.5 0.092 52.0 0.119 60.0 0.119 53.8

172 Results and Discussion

40 (mg/l) 50 (mg/l) 75 (mg/l) 0.18 100 (mg/l) 150 (mg/l) 0.16

0.14

) 0.12

nm 0.1 600

. ( 0.08 D .

O 0.06

0.04

0.02

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (63): Growth of strain MAM- 78 on different concentrations of anthracene.

80 40 (mg/l) 50 (mg/l) 75 (mg/l) 100 (mg/l) 150 (mg/l) 70

60

50

40

30 Protein (ug/ml)Protein

20

10

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (64): Extracellular protein of strain MAM-78 on different concentrations of anthracene.

173 Results and Discussion

Table (34): Growth and extracellular protein of strain E. cloacae MAM-4 on different concentrations of anthracene.

Conc. 40 50 75 100 150 Incubation ( mg/l) (mg/l) (mg/l) ( mg/l) (mg/l) period(days) Protein Protein Protein Protein Protein OD OD OD OD OD Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.140 31.5 0.155 29.7 0.146 31.5 0.151 33.7 0.167 30.6 1 0.148 32.6 0.152 31.5 0.164 40.0 0.160 40.0 0.184 40.0 2 0.164 23.0 0.164 15.4 0.170 43.8 0.182 12.3 0.186 17.7 3 0.156 19.2 0.152 15.3 0.158 20.0 0.166 23.8 0.196 30.7 4 0.157 20.0 0.160 18.0 0.159 23.0 0.174 24.0 0.200 28.0 5 0.158 21.5 0.163 21.5 0.169 25.4 0.181 26.2 0.208 26.2 6 0.157 26.9 0.160 27.4 0.166 35.0 0.177 30.6 0.200 30.0 7 0.153 33.7 0.155 30.9 0.165 38.9 0.175 32.8 0.195 33.7 14 0.151 39.9 0.153 33.0 0.165 44.7 0.175 38.4 0.188 38.5 21 0.156 40.8 0.154 34.6 0.138 50.0 0.153 41.5 0.177 40.0

174 Results and Discussion

40 (mg/l) 50 (mg/l) 75 (mg/l) 0.25 100 (mg/l) 150 (mg/l)

0.2 ) 0.15 nm 600 . ( D

. 0.1 O

0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (65): Growth of strain E. cloacae MAM -4 on different concentrations of anthracene.

60 40 (mg/l) 50 (mg/l) 75 (mg/l) 100 (mg/l) 150 (mg/l) 50

40

30 Protein(ug/ml) 20

10

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (66): Extracellular protein of strain E.cloacae MAM-4 on different concentrations of anthracene.

175 Results and Discussion

So, we try to grow some of the isolated strains on higher concentrations of anthracene (200, 300 and 400 mg/L) to determine the abilities of these isolates to grow and use the higher concentrations of anthracene as a sole carbon and energy source and know how the growth profiles of these strains will be. The growth of the isolated strain MAM-26 on higher concentrations of Anth. was indicated in Table (35) and Figure (67).

The growth showed increased growth over the initial till the end of the incubation period 200 mg/L Anth. while the increase continue till the fifth day at concentrations 300 and 400 mg/L anth. Also, till the end of the incubation period (21 days) the growth was more than the initial. Another observation, that the growth was concentration dependent i.e., the more increase in concentration of Anth. the more increase in growth. The extracellular protein produced by the isolated strain MAM-26 grown on higher concentrations of Anth. as in Table (35) and Figure (68) revealed that protein was flactuated between increase and decease a long the whole incubation period.

Growth profile of isolated strain MAM-29 on higher concentrations of Anth. was indicated in Table (36) and Figure (69). From the results, it was clear that MAM-29 growth increased at all the three concentrations used till the fifth day and then began to decrease gradually. Also the growth profile indicated that growth was concentration dependent. The extracellular protein of MAM-29 as shown in Table (36) and Figure (70) cleared that extracellular protein secretion showed increased over the initial till the forth day gradually then began to decrease till the sixth day and began to increase till the seventh day then deceased i.e. flactuated.

176 Results and Discussion

Table (35): Growth and extracellular protein of strain MAM-26 on higher concentrations of anthracene. Conc.

200 300 400

(mg/l) (mg/l) (mg/l) Incubation period(days) Protein Protein Protein OD OD OD Zero (ug/ml) (ug/ml) (ug/ml) time 0.163 34.6 0.185 35.0 0.193 34.5

1 0.177 33.8 0.232 46.9 0.248 32.0

2 0.200 42.3 0.214 33.8 0.250 34.6

3 0.230 40.0 0.240 35.0 0.280 36.0

4 0.246 39.2 0.257 40.8 0.322 39.2

5 0.244 48.5 0.281 40.8 0.352 42.3

6 0.232 43.8 0.278 31.5 0.337 30.8

7 0.216 46.9 0.252 41.5 0.320 36.9

14 0.204 40.0 0.252 40.0 0.286 36.8

21 0.185 38.5 0.242 38.5 0.276 36.2

177 Results and Discussion

0.45 0.4 200 mg/l 300 mg/l 400 mg/l 0.35 0.3 ) 0.25 nm

600 0.2 .( D .

O 0.15 0.1 0.05 0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (67): Growth of strain MAM-26 on higher concentrations of anthracene.

70 200 mg/l 300 mg/l 400 mg/l 60

50

40

30

Protein (ug/ml) 20

10

0 0 1 2 3 4 5 6 7 14 21

Incubation period (days)

Figure (68): Extracellular protein of strain MAM-26 on higher concentrations of anthracene.

178 Results and Discussion

Table (36): Growth and extracellular protein of strain MAM-29 on higher concentrations of anthracene.

Conc.

200 300 400

(mg/l) (mg/l) (mg/l) Incubation period(days) Protein Protein Protein OD OD OD Zero (ug/ml) (ug/ml) (ug/ml) time 0.395 30.0 0.422 36.0 0.412 34.0 1 0.419 40.7 0.480 59.2 0.475 46.9 2 0.419 52.0 0.456 63.0 0.462 60.8 3 0.420 60.0 0.440 65.0 0.470 62.0 4 0.412 70.0 0.448 70.7 0.441 63.8 5 0.435 63.8 0.449 69.2 0.495 62.3 6 0.396 65.4 0.428 57.7 0.448 58.5 7 0.378 50.8 0.404 65.4 0.424 72.3 14 0.360 51.2 0.388 64.0 0.384 68.0 21 0.338 53.8 0.362 63.0 0.373 62.3

179 Results and Discussion

0.7 200 mg/l 300 mg/l 400 mg/l 0.6

0.5 ) 0.4 nm 600 .(

D 0.3 . O 0.2

0.1

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (69): Growth of strain MAM-29 on higher concentrations of anthracene.

100 200 mg/l 300 mg/l 400 mg/l 90 80 70 60 50 40 Protein (ug/ml)Protein 30 20 10 0 0 1 2 3 4 5 6 7 14 21

Incubation period (days)

Figure (70): Extracellular protein of strain MAM-29 on higher concentrations of anthracene.

180 Results and Discussion

The growth of the isolated strain MAM-62 on higher concentration of Anth. as shown in Table (37) and Figure (71) indicated that the maximum growth was recorded after 24 hours, then the growth began to decrease a long the whole of the rested incubation period.

The extracellular protein secreted by the isolate MAM-62 as shown in Table (37) and Figure (72) indcated that protein production of 200 and 300 mg/L Anth. increased from the beginning till the second day, then decreased slightly till the end of the incubation period except of the 5th day which show exceptional increase in protein production . However in case of 400 mg/L Anth. the increase in protein was continued till the end of incubation period.

In case of isolated strain MAM-68, the growth was decreased gradually from the beginning at concentrations (200 and 300 mg/L) Anth., but at 400 mg/L Anth., growth increased for 24 hours and began to decrease gradually as in Table (38) and Figure (73).

Extracellular protein production by MAM-68 was shown in Table (38) and Figure (74). The results indicated that, there was an increase in protein secretion at the beginning and continue till the fifth day then began to decrease at the sixth day and increased again at the seventh day and became stable till the end of the incubation period at concentrations 200 and 300 mg/L Anth. However, at 400 mg/L, after the seventh day of incubation extracellular protein began to decrease.

181 Results and Discussion

Table (37): Growth and extracellular protein of strain MAM-62 on higher concentrations of anthracene.

Conc.

200 300 400

(mg/l) (mg/l) (mg/l) Incubation period(days) Protein Protein Protein OD OD OD Zero (ug/ml) (ug/ml) (ug/ml) time 0.422 50.8 0.446 50.2 0.465 50.0

1 0.452 62.3 0.475 64.6 0.482 52.3

2 0.396 65.4 0.465 88.5 0.470 66.9

3 0.400 64.0 0.460 85.0 0.450 70.0

4 0.402 63.8 0.461 80.0 0.449 76.2

5 0.383 76.7 0.440 82.3 0.473 73.8

6 0.386 65.4 0.430 70.0 0.452 72.3

7 0.347 66.9 0.422 78.5 0.438 70.0

14 0.337 60.0 0.430 70.0 0.390 72.0

21 0.305 63.8 0.355 66.2 0.367 74.6

182 Results and Discussion

0.6 200 mg/l 300 mg/l 400 mg/l

0.5

) 0.4 nm 0.3 600 .( D . O 0.2

0.1

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (71): Growth of strain MAM- 62 on higher concentrations of anthracene.

200 mg/l 300 mg/l 400 mg/l 100

80

60

40 Protein Protein (ug/ml)

20

0 0 1 2 3 4 5 6 7 14 21

Incubation period (days)

Figure (72): Extracellular protein of strain MAM-62 on higher concentrations of anthracene.

183 Results and Discussion

Table (38): Growth and extracellular protein of strain MAM-68 on higher concentrations of anthracene.

Conc.

200 300 400 Incubation (mg/l) (mg/l) (mg/l) period(days) Protein Protein Protein OD OD OD Zero (ug/ml) (ug/ml) (ug/ml) time 0.447 46.2 0.427 48.0 0.410 42.0

1 0.430 64.6 0.385 56.2 0.456 54.6

2 0.375 66.2 0.368 63.0 0.422 72.3

3 0.375 67.0 0.367 66.0 0.425 70.0

4 0.378 67.7 0.373 69.2 0.426 64.6

5 0.375 67.7 0.352 70.8 0.419 71.5

6 0.397 58.5 0.377 62.3 0.421 64.6

7 0.340 65.4 0.374 67.7 0.405 70.8

14 0.321 64.8 0.330 67.8 0.358 60.0

21 0.303 64.6 0.298 67.7 0.356 50.0

184 Results and Discussion

0.5 200 mg/l 300 mg/l 400 mg/l 0.45 0.4 0.35

) 0.3

nm 0.25 600

. ( 0.2 D .

O 0.15 0.1 0.05 0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (73): Growth of strain MAM-68 on higher concentrations of anthracene.

120 200 mg/l 300 mg/l 400 mg/l 100

80

60

Protein (ug/ml) 40

20

0 0 1 2 3 4 5 6 7 14 21

Incubation period (days)

Figure (74): Extracellular protein of strain MAM-68 on higher concentrations of anthracene.

185 Results and Discussion

The growth profile of the isolated strain Enterobacter cloacae MAM-4 on higher concentrations of Anth. as indicated in Table (39) and Figure (75) revealed that the growth at 200 and 300 mg/L Anth. was flactuated increase flowed by decrease, but in case of 400 mg/L Anth. there was a sudden increase between the first and second days followed by gradual decrease along the incubation period. The results also revealed that growth was concentration dependent.

The extracellular protein of MAM-4 indicated that sudden increase was recorded after 24 hours incubation for concentrations 300 mg/L Anth. followed by decrease at the second day followed by increase as in Table (39) and Figure (76).

The count of the different isolates after 21 days incubation on different concentrations of Anth. had been indicated in Table (40). The results revealed that the best isolate in count after 21 days incubation at different concentrations was isolate MAM-26. The initial count was 5.0x107 CFU/ml, this count became 18.0x107, 15.0x107, 12.0x107, 10.0x107, and 8.0 x107CFU/ml on 40, 50, 75, 100, and 150 mg/L Anth. respectively.

Meanwhile, all the tested strains on the three higher anthracene (Anth.) concentrations, their counts were higher than their initial counts. Except concentration 200 and 300 mg/L of strain MAM-68 which was lower in count than the intial. The best strain in growing on Anth. at higher concentrations was MAM-29 its initial count was 3.2x107 CFU/ml, this count became 64.1x107, 55.7 x107 and 71.0 x107 CFU/ml after 21 days on 200, 300, 400 mg/L Anth. respectively as in Table (40).

186 Results and Discussion

Table (39): Growth and extracellular protein of strain E.cloacae MAM- 4 on higher concentrations of anthracene.

Conc.

200 300 400

(mg/l) (mg/l) (mg/l) Incubation period(days) Protein Protein Protein OD OD OD Zero (ug/ml) (ug/ml) (ug/ml) time 0.115 25.4 0.124 28.0 0.135 27.0

1 0.181 30.0 0.159 65.4 0.139 44.6

2 0.138 33.0 0.138 30.8 0.234 42.3

3 0.140 35.0 0.160 34.9 0.225 44.0

4 0.172 38.5 0.182 43.8 0.216 48.5

5 0.141 45.4 0.162 44.6 0.200 50.8

6 0.133 37.7 0.180 40.8 0.191 47.7

7 0.133 43.0 0.150 46.2 0.170 54.6

14 0.104 44.0 0.159 44.0 0.131 50.0

21 0.092 45.4 0.136 41.5 0.148 43.0

187 Results and Discussion

0.25 200 mg/l 300 mg/l 400 mg/l

0.2

0.15 ) nm

600 0.1 . ( . D . O 0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (75): Growth of strain E.cloacae MAM-4 on higher concentrations of anthracene.

70 200 mg/l 300 mg/l 400 mg/l 60

50

40

30

Protein (ug/l) 20

10

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (76): Extracellular protein of strain E.cloacae MAM-4 on higher concentrations of anthracene.

188 Results and Discussion

The results of degradation of anthracene as determined by HPLC revealed that the best anthracene degrader was isolate MAM-62 as indicated from Table (41) and Figure (77). Isolate MAM-62 degraded 85% of 150 mg/L Anth. This strain also degraded 94% of 40 mg/L Anth. also.

The above results was confirmed by other investigators and it may be also exceed the results which they found as follow:

Loser et al. (2004), found that the fastest and most extensive PAHs degradation during compositing occurred at 30C and the higher temperatures inhibited the degradation of anthracene and pyrene.

Exploration on co-metabolism showed that the highest degradation efficiency was reached at equal concentration of lactose and anthracene. Excessive carbon source would actually hamper the degradation was efficiency (Ye et al., 2011).

Anthracene (AC) is hydroxylated at first and converted into anthraquinone which is in turn hydroxylated further to form 1-4 dihydroxyanthraquinone. Cladosporium herbarum was able to degrade AC during the first 24 h but this metabolization probably to a toxic compound for this fungus, explaining the strong decrease in degradation rate after 48 and 72 h incubation (Guiraud et al., 2008).

189 Results and Discussion

Table (40): Count of the selected isolated strains on different concentrations of anthracene after 21 days incubation period.

Concentrations (mg/L) e

Initial count Initial 40 50 75 100 150 200 300 400 (CFU/ml) IO count(CFU/ml)

Isolatecod count logN count logN count logN count logN count logN count logN count logN count logN count logN count logN

MAM- 5.0*107 7.69 18.0*107 8.25 15.0*107 8.17 12.0*108 8.0 1.0*107 7.0 8.0*107 7.90 13.0*107 8.11 47.2*107 8.67 55.0*107 8.74 41.2*107 8.61 26

MAM- 7 7 7 7 7 7 7 7 7 7 7.0*10 7.84 1.5*10 7.17 3.0*10 7.47 2.6*10 7.41 4.0*10 7.6 7.0*10 7.84 3.2*10 7.50 64.1*10 8.80 55.7*10 8.74 71.0*10 8.85 29

MAM- 7 7 7 7 7 7 34.0*10 8.53 3.2*10 7.50 3.0*10 6.47 3.0*10 7.47 9.0*10 7.9 0.4*10 6.60 N.D. ** N.D. ** N.D. ** N.D. ** 43

MAM- 27.0*107 8.43 7.0*107 7.84 8.0*107 7.90 7.0*107 7.84 9.0*107 7.95 16.0*107 8.20 20.0*107 8.30 48.0*107 8.68 46.5*107 8.66 32.0*107 8.50 62

MAM- 3.0*107 7.47 2.5*107 7.39 3.0*107 7.47 5.5*107 7.74 4.0*107 7.60 4.4*107 7.64 42.0*107 8.62 200.0*107 8.30 22.0*107 8.34 85.0*107 8.92 68

MAM- 8.0*107 7.90 12.0*107 8.07 10.0*107 8.0 5.0*107 7.69 8.0*107 7.90 7.0*107 7.84 N.D. ** N.D. ** N.D. ** N.D. ** 78

MAM- E. 5.0*106 6.69 1.0*107 7.00 2.0*107 7.30 1.0*107 7.00 1.3*107 7.11 1.0*107 7.00 2.0*107 7.30 5.4*107 7.73 10.7*107 8.02 15.0*107 8.17 cloacae- 4

190 Results and Discussion

Table (41): Degradation percentage of anthracene after 21 days by HPLC.

Degradation % Isolate code 40 (mg/l) 50 (mg/l) 75 (mg/l) 100 (mg/l) 150 (mg/l) MAM-26 42.5% 49% 39.5% 31.5% 11% MAM-29 74% 67% 63% 53.5% 31% MAM-43 80.5% 96% 62% 78.5% 79% MAM-62 94% 73% 74.5% 39.5% 85% MAM-68 59% 32% 45.5% 19% 36.5% MAM-78 52.5% 75% 50% 39% 29.5%

MAM-E. 71% 56% 43.5% 45% 39% cloacae *count = CFU/ml

MAM-26 MAM-29 MAM-43 MAM-62 MAM-68 MAM-78 MAM-E. cloacae

120%

100%

80%

60%

Degradation % Degradation 40%

20%

0% 40 50 75 100 150 Concentration (mg/L)

Figure (77): Degradation percentage of anthracene after 21 days by HPLC.

191 Results and Discussion

Brodkorb and Legge (1992) added a tar-like oil contaminated soil to liquid cultures of Phanerochaete chrysosporium, and reported 37.7% mineralization of anthracene compared to 19.5% mineralization by native microflora after 21 days at 37C.

3.7. Growth and degradation of pyrene by the most potent isolated strains.

Growth of the isolated strain MAM-26 on different concentrations of Pyr. as indicated in Table (42) and Figure (78) revealed that there was good growth on the lowest concentration (100 g/L) Pyr. at the third day then decreased gradually. The concentration (200 g/l) of Pyr. indicated an increase to reach maximum at sixth day. At higher concentrations (300 and 400 g/L) of Pyr., the highest growth was recorded at the forth day. Meanwhile, 500 g/L Pyr. Showed high growth at the second and seventh days.

Extracellular protein secreted by MAM-26 grown on different concentration of Pyr. showed a flactual increase and decrease during the time of incubation i.e. (flactuated) as indicated by Table (42) and Figure (79).

Growth profile of isolate MAM-29 was cleared in Table (43) and Figure (80). The low concentrations (100, 200 and 300 g/L) Pyr. reached their maximum growth after 24 hours incubation, while at higher concentrations (400 and 500 g/L) Pyr. it need more time to reach maximum growth at the forth day.

192 Results and Discussion

Table (42): Growth and extracellular protein of strain MAM-26 on different concentrations of pyrene.

Conc. 100 200 300 400 500 Incubation (ug/l) (ug/l) (ug/l) (ug/l) (ug/l) period(days) Protein Protein Protein Protein Protein Zero O.D. O.D. O.D. O.D. O.D. (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) time 0.164 32.0 0.108 6.0 0.164 20.0 0.144 10.0 0.142 25.0 1 0.208 25.4 0.202 7.8 0.203 56.1 0.220 12.3 0.186 33.8 2 0.192 48.0 0.145 30.0 0.132 40.8 0.103 53.0 0.135 63.0 3 0.700 45.0 0.160 36.0 0.200 36.9 0.600 44.0 0.137 55.0 4 0.400 37.0 0.165 40.2 0.260 32.0 0.700 30.0 0.140 23.0 5 0.149 26.1 0.173 47.7 0.137 30.8 0.193 12.3 0.143 11.5 6 0.137 35.0 0.282 53.0 0.136 73.0 0.124 32.3 0.130 90.0 7 0.114 44.6 0.133 25.3 0.140 51.6 0.109 35.4 0.185 66.9 14 0.136 35.0 0.167 30.0 0.172 7.0 0.118 20.0 0.165 40.0 21 0.138 110 0.140 40.8 0.159 92.3 0.131 15.4 0.114 32.3

193 Results and Discussion

100 (ug/l) 200 (ug/l) 300 (ug/l) 0.8 400 (ug/l) 500 (ug/l)

0.7

0.6

) 0.5 nm 0.4 600 . ( D . 0.3 O

0.2

0.1

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (78): Growth of strain MAM-26 on different concentrations of pyrene.

120 100 (ug/l) 200 (ug/l) 300 (ug/l) 400 (ug/l) 500 (ug/l) 100

80

60

Protein (ug/ml) Protein 40

20

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (79): Extracellular protein of strain MAM-26 on different concentrations of pyrene

194 Results and Discussion

The extracellular protein produced by MAM-29 as shown in Table (43) and Figure (81) indicated that there was sudden increase at the first and second days followed by flactuation increase and decrease pattern.

Growth profile of isolate MAM-62 on the five concentrations of Pyr. indicated that the highest growth recorded after 24 hours and then began to decrease along the rest period of incubation as shown in Table (44) and Figure (82).

Extracellular protein produced by MAM-62 showed an increase at the first and second days followed by gradual decrease to reach the lowest production at fifth day, followed by increase till the end of the incubation period for the five concentrations used as shown in Table (44) and Figure (83).

Table (45) and Figure (84) indicated that growth of isolate MAM-68 on the five concentrations of Pyr. reached their maximum growth after 24 hours incubation, then began to decrease. The extracellular protein produced by MAM-68 increased at the first and second days followed by flactuation as shown in Table (45) and Figure (85).

195 Results and Discussion

Table (43): Growth and extracellular protein of strain MAM-29 on different concentrations of pyrene.

Conc.

100 (ug/l) 200 (ug/l) 300 (ug/l) 400 (ug/l) 500 (ug/l) Incubation period(days) Protein Protein Protein Protein Protein Zero O.D. O.D. O.D. O.D. O.D. (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) time 0.131 17.6 0.210 19.6 0.110 15.6 0.171 12.4 0.155 10.2 1 0.273 43.6 0.215 43.0 0.232 44.1 0.269 92.3 0.239 60.8 2 0.100 53.8 0.110 63.8 0.202 87.7 0.219 59.2 0.198 70.0 3 0.170 52.0 0.108 61.0 0.170 60.8 0.600 55.8 0.300 60.0 4 0.173 51.6 0.107 60.0 0.160 55.9 0.690 49.0 0.450 44.0 5 0.176 50.8 0.106 59.2 0.153 40.0 0.195 48.5 0. 147 31.5 6 0.117 63.8 0.146 100.7 0.141 69.2 0.194 123.0 0.188 121.5 7 0.127 63.8 0.092 57.7 0.107 66.1 0.109 73.8 0.160 66.9 14 0.110 77.0 0.179 77.0 0.203 84.8 0.127 74.0 0.155 78.9 21 0.172 136.0 0.189 120.0 0.185 100.0 0.169 84.6 0.185 96.9

196 Results and Discussion

100 (ug/l) 200 (ug/l) 300 (ug/l) 0.8 400 (ug/l) 500 (ug/l) 0.7

0.6 ) 0.5 nm 0.4 600 . ( D . 0.3 O

0.2

0.1

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (80): Growth of strain MAM-29 on different concentrations of pyrene.

160 100 (ug/l) 200 (ug/l) 300 (ug/l)

140 400 (ug/l) 500 (ug/l)

120

100

80

60 Protein (ug/ml)

40

20

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (81): Extracellular protein of strain MAM-29 on different concentrations of pyrene.

197 Results and Discussion

Table (44): Growth and extracellular protein of strain MAM-62 on different concentrations of pyrene.

Conc. 100 200 300 400 500 Incubation (ug/l) (ug/l) (ug/l) (ug/l) (ug/l) period(days) Protein Protein Protein Protein Protein Zero O.D. O.D. O.D. O.D. O.D. (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) time 0.197 38.3 0.124 38.3 0.120 39.7 0.126 40.1 0.190 37.4 1 0.335 88.1 0.362 75.3 0.353 85.4 0.361 56.1 0.286 59.2 2 0.174 75.4 0.161 78.5 0.183 84.6 0.164 86.2 0.199 91.5 3 0.180 65.9 0.170 69.4 0.180 74.3 0.170 79.3 0.200 83.2 4 0.190 54.7 0.180 54.5 0.179 64.9 0.197 64.2 0.210 74.1 5 0.218 44.6 0.196 48.5 0.179 51.5 0.235 53.0 0.236 58.5 6 0.214 56.1 0.204 64.6 0.171 97.7 0.155 72.3 0.162 64.6 7 0.143 61.3 0.203 93.0 0.212 136.2 0.194 107.7 0.133 118.5 14 0.218 88.9 0.133 100.7 0.185 140.0 0.164 100.9 0.153 122.8 21 0.133 136.9 0.092 136.9 0.123 148.5 0.102 111.5 0.150 154.6

198 Results and Discussion

100 (ug/l) 200 (ug/l) 300 (ug/l) 0.4 400 (ug/l) 500 (ug/l) 0.35

0.3

) 0.25 nm 0.2 600 . ( . D . 0.15 O

0.1

0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (82): Growth of strain MAM- 62 on different concentrations of pyrene.

180 100 (ug/l) 200 (ug/l) 300 (ug/l) 160 400 (ug/l) 500 (ug/l)

140

120

100

80 Protein (ug/ml) 60

40

20

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (83): Extracellular protein of strain MAM-62 on different concentrations of pyrene.

199 Results and Discussion

From the results of Table (46) and Figure (86) it was clear that growth of Enterobacter cloacae MAM-4 on the five concentrations of Pyr. reached the maximum growth after 24 hours incubation period followed by significant decrease.

Profile of extracellular protein secreted by E. cloacae MAM-4 when grown on different concentrations of Pyr. indicated an increase in protein production at the first and second days followed by decrease to reach the lowest production at fifth day followed again by increase at the sixth day (i.e. flactuated) as shown in Table (46) and Figure (87).

The count of the isolated strains on the five concentrations of Pyr. as indicated in Table (47) revealed that, the initial counts were ranging from 2.5x107 CFU/ml to 6.0 x107 CFU/ml. However the counts of these strains were ranging from 1.4x106 CFU/ml to 5.4x107 CFU/ml at the end of incubation period (21 days).

These results, cleared that the count of different strains may be decrease in number than the initial count, this may be explain on the bases that they enter the decline phase. However, isolated strain MAM-29 recorded the same count 2.7x107 CFU/ml from the begining to the end of the incubation period at concentration 500µg/L.

The degradation percentage of pyrene by the different isolated strains was indincated in Table (48) and Figure (88).

The results revealed that MAM-62 and MAM-26 were the best pyrene degraders. They degraded 94.1% and 91.9% from 100µg/L and 51.4% and 59.4% from 500µg/L pyrene respectively.

The above results have been confirmed by the results of other investigators as the following.

200 Results and Discussion

Table (45): Growth and extracellular protein of strain MAM-68 on different concentrations of pyrene. Conc. 100 200 300 400 500 Incubation (ug/l) (ug/l) (ug/l) (ug/l) (ug/l) period(days) Protein Protein Protein Protein Protein Zero O.D. O.D. O.D. O.D. O.D. (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) time 0.184 40.7 0.149 44.8 0.191 38.9 0.177 50.0 0.162 44.9 1 0.283 51.5 0.260 93.8 0.220 55.3 0.299 87.6 0.306 86.9 2 0.127 71.5 0.197 80.7 0.122 112.3 0.179 75.4 0.186 73.0 3 0.160 69.0 0.194 75.0 0.126 100.0 0.179 70.0 0.180 60.0 4 0.172 66.0 0.187 70.0 0.170 80.0 0.177 65.4 0.170 50.0 5 0.186 81.5 0.186 61.5 0.184 53.0 0.177 59.2 0.167 41.5 6 0.176 106.2 0.169 91.5 0.098 103.0 0.126 56.9 0.110 104.6 7 0.094 74.6 0.078 80.0 0.176 73.0 0.165 68.5 0.170 79.2 14 0.183 88.0 0.105 75.0 0.165 68.0 0.155 77.0 0.131 70.0 21 0.156 100.7 0.176 72.0 0.170 62.3 0.155 125.4 0.146 61.5

201 Results and Discussion

0.35 100 (ug/l) 200 (ug/l) 300 (ug/l) 400 (ug/l) 500 (ug/l) 0.3

0.25 )

nm 0.2 600

. ( 0.15 D . O 0.1

0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (84): Growth of strain MAM-68 on different concentrations of pyrene.

140 100 (ug/l) 200 (ug/l) 300 (ug/l) 400 (ug/l) 500 (ug/l) 120

100

80

60 Protein (ug/ml)Protein 40

20

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (85): Extracellular protein of strain MAM-68 on different concentrations of pyrene.

202 Results and Discussion

Table (46): Growth and extracellular protein of strain E.cloacae MAM-4 on different concentrations of pyrene. Conc. 100 200 300 400 500 Incubation (ug/l) (ug/l) (ug/l) (ug/l) (ug/l) period(days) Protein Protein Protein Protein Protein Zero O.D. O.D. O.D. O.D. O.D. (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) time 0.146 10.0 0.132 11.0 0.157 13.8 0.106 9.0 0.121 12.0 1 0.304 40.0 0.250 17.7 0.181 19.2 0.233 30.0 0.241 34.6 2 0.130 40.0 0.137 36.9 0.188 37.0 0.143 70.7 0.176 66.9 3 0.135 40.0 0.142 33.0 0.170 32.0 0.138 60.0 0.170 44.0 4 0.140 39.0 0.148 32.0 0.155 28.0 0.130 44.0 0.150 32.0 5 0.151 38.5 0.152 32.0 0.126 26.2 0.129 30.7 0.148 26.9 6 0.154 79.2 0.179 54.6 0.036 86.9 0.026 107.6 0.167 102.3 7 0.106 136.9 0.034 45.4 0.020 54.2 0.037 53.8 0.148 56.2 14 0.113 80.0 0.105 40.0 0.110 80.0 0.081 40.0 0.156 80.0 21 0.140 40.0 0.124 23.0 0.140 110.7 0.155 22.3 0.114 130.8

203 Results and Discussion

0.35 100 (ug/l) 200 (ug/l) 300 (ug/l)

400 (ug/l) 500 (ug/l) 0.3

0.25 )

nm 0.2 600

. ( 0.15 D . O 0.1

0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (86): Growth of strain E.cloacae MAM-4 on different concentrations of pyrene.

160 100 (ug/l) 200 (ug/l) 300 (ug/l)

140 400 (ug/l) 500 (ug/l)

120

100

80

60 Protein (ug/ml)Protein

40

20

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (87): Extracellular protein of strain E.cloacae MAM-4 on different concentrations of pyrene.

204 Results and Discussion

Table (47): Count of the selected isolated strains on different concentrations of pyrene after 21 days incubation.

Concentrations (ug/L) Isolate Initial count 100 200 300 400 500 code (CFU/ml) count logN count logN count logN count logN count logN count logN

MAM-26 2.9*107 7.46 3.0*107 7.47 2.6*107 7.41 1.7*107 7.23 4.0*106 6.60 9.0*106 6.95

MAM-29 2.7*107 7.43 5.0*106 6.69 1.4*106 6.14 2.5*106 6.39 5.4*107 7.73 2.7*107 7.43

MAM-62 2.5*107 7.39 6.6*106 6.81 1.1*107 7.04 1.0*107 7.00 2.2*107 7.34 6.0*106 6.77

MAM-68 6.0*107 7.77 1.6*107 7.20 4.5*107 7.65 1.7*107 7.23 3.3*107 7.51 1.7*107 7.23 E. cloacae 2.8*107 7.44 1.0*107 7.00 1.9*107 7.27 3.0*107 7.47 3.0*107 7.47 1.2*107 7.07 MAM-4

205 Results and Discussion

In mineral salt medium, Mycobacterium sp. mineralized 50% of 250g/ml of pyrene in 2 to 3 days (Jiménez and Bartha, 1996).

The native sediment microorganisms have shown 35% Pyrene degradation (Ravelet et al., 2001).

Phenanthrene dissipated more rapidly and completely than pyrene, which is in agreement with many other studies, suggesting that high- molecular-weight PAHs are more resistant to microbial attack than low- molecular weight PAHs (Tabak et al., 2003).

Feitkenhauer et al. (2003) found that higher mass transfer rates and PAH solubilities and hence bioavailabilites can be obtained at higher temperatures. Mixed and pure cultures of aerobic and extreme thermophilic microorganisms (Bacillus Spp., Thermus sp.) were used to degrade PAH compounds and PAH/alkane mixtures at 65C. Optimal growth temperatures were in the range of 60-70C at pH values of 6-7. the conversion of PAH with 3-5 rings (acenaphthene, fluoranthene, pyrene, benzo[e]pyrene) was demonstrated.

Ho et al. (2000) isolated fluoranthene and pyrene-degrading strains by liquid culture enrichment; 19 of 21 pyrene-degrading strains were gram positive, and 7 of these were Mycobacteria. A total of 28 fluroanthene- degrading strains were all gram negative, and 4 of them belonged to the genus Sphingomonas.

Ravelet et al. (2001) found that a supply of acted as an inhibitor to pyrene disappearance.

206 Results and Discussion

Pyrene was utilized by B. pumilus strains 96-1, 96-6, 96-7, 28-11 and 212-1, and phenanthrene was utilized as sole carbon source by strains of B. pumilus, B. subtitis and M. Luteus strains. Naphthalene was utilized by all the selected strains (B. pumilus, B. subtitis, M. luteus, A. faecalis and Enterobacter sp.), whereas fluroanthene was only utilized by one strain affiliated to A. faecalis. Pyrene-removing bacteria were only found in the genus Bacillus, a microbial group that also showed an increased phenanthrene-removal capacity (Toledo et al., 2006).

Complete biodegradation of pyrene took longer time than that for fluoranthene and phenanthrene. (Yu et al., 2005a).

At the end of 4th week, natural attenuation based on the presence of autochthonous microorganisms degraded more than 99% fluoranthene and Phen. but only around 30% of Pyrene were degraded (Yu et al., 2005a), they studied the biodegrdatation of a mixture of fluorene, phenanthrene and pyrene by a bacterial consortium made up of three strains of Rhodococcus sp., Acineto-bacter sp and Pseudomonas sp. Their results showed that the addition of his consortium into sediments significantly enhanced the efficiency of fluorene and phenanthrene biodegrdation but not that of pyrene. After 2 weeks of incubation the removal rate reached the values of 97% and 99% for phenanthrene and fluorine, respectively. However, only about 10% of pyrene was degraded.

However, PAHs of more than 3 benzene rings remained almost unchanged (Wang et al., 2007).

These results indicate the pyrene was generally more stable, recalcitrant, and more difficult to be removed by microlagae (Lei et al., 2007).

207 Results and Discussion

In the present study, the presence of fluoranthene was found to stimulate the removal of pyrene and vice versa, suggesting some positive interaction might occur when these two PAHs were mixed together.

It is consistent with the fact that pyrene has four aromatic rings while phenanthrene has three. PAHs with more aromatic rings are usually more resistant to degradation than those with fewer aromatic rings (Doyle et al., 2008).

PAH-degrading microbial consortium and its pyrene-degrading plasmids were enriched from the sediment samples of Huian mangroves. The consortium YL showed degrading abilities of 92.1%, and 95.8% of pyrene and fluroanthene at 50 mg1-1 after 21 days incubation (Lin and Cai, 2008) respectively.

Anastasi et al. (2009) evaluated the potential of a consortium of three basidiomycetes isolated from compost for pyrene degradation in sterile soil microcosms; the basidiomycetes were able to efficiently colonize soil and remove about 56% of the pyrene in 28 days.

208 Results and Discussion

Table (48): Degrdation percentage of Pyrene after 21 days by HPLC.

% degrdatation Isolate code 100g/L 200g/L 300g/L 400g/L 500g/L

MAM-26 91.9% 89.3% 90.0% 75.8% 59.4%

MAM-29 95.0% 90.5% 90.3% 70.1% 50.7%

MAM-62 94.1% 90.8% 90.6% 72.9% 51.4%

MAM-68 77.3% 70.1% 55.4% 17.9% 9.9%

E.cloacae 76.5% 68.2% 45.2% 45.9% 11.5% MAM-4

MAM-26 MAM-29 MAM-43 MAM-62 MAM-68 MAM-78 MAM-E. cloacae

100%

90%

80%

70%

60%

50%

40% Degradation % Degradation 30%

20%

10%

0% 100 200 300 400 500 Concentration (ug/l)

Figure (88): Degrdation percentage of Pyrene after 21 days by HPLC.

209 Results and Discussion

It is well known that very few organisms are able to degrade a single compound completely (Li et al., 2008), Bacillus cereus Py5 and Bacillus megaterium Py6 were isolated from the consortium and observed consuming 65.8% and 33.7% of pyrene (50 mg/l) within three weeks, respectively.

The enriched Escherichia coli DH5a cells containing the plasmids of YL were demonstrated to degrade 85.7% of the original pyrene concentration at the 21st day (Lin and Cai, 2008).

HPLC analysis showed that the degradation rate of pyrene 5 mg/L by the endophytic bacterial strain 12J1 was 83.8% under 28C for 7 days (Sheng et al., 2008).

Biodegrdation of pyrene by Mycobacterium frederiksbergense was studied in two phase partitioning bioreactor (TPPB). The TPPB achieved complete biodegradation of pyrene, and during the active degradation phase utilization rates of 270, 230, 139, 82 mg/L/d. for intitial pyrene loading concentrations of 1000, 600, 400 and 200 mg/L (Mahanty et al., 2008).

3.8. Growth and degradation of benzo-a-anthracene by the most potent isolated strains.

The degradation rate constants of both phenanthrene and pyrene increased with the PAH concentration in the cultures containing 2 to 50 mg /l PAHs. However, at the level of 100 mg/l, the degradation rate constants were the lowest because the organism failed to degrade the remaining PAHs after 6 d. It appears that the organism entered an inactive phase after 6 d incubation in the liquid culture (Ting et al., 2011).

210 Results and Discussion

On contrast to pyrene, growth on Benzo-a-anthracene (B-a-Anth.) revealed no or slightly growth on B-a-Anth. Isolate MAM-26 showed slight increase on first or second days at low concentrations (100 and 200 g/L) B- a-Anth. At 300 g/L, the maximum growth was reached at the third day as indicated in Table (49) and Figure (89). At higher concentrations (400 and 500 g/L) B-a-Anth. first day revealed the best growth even it was slight. Extracellular protein produced by MAM-26 indicated that the highest protein production was recorded at the third day for all concentrations then decreased as shown in Table (49) and Figure (90).

Growth profile of isolate MAM-29 on different concentrations of B-a-Anth. was indicated in Table (50) and Figure (91). Except the low concentration, 100g/L, all the four concentrations revealed increase in growth after 24 hours, then began to decrease. The highest extracellular protein produced by MAM-29 on the five concentrations of B-a-Anth was recorded at the third day as show in Table (50) and Figure (92). After that protein production decreased sharply till the fifth day.

Growth profile of isolate MAM-62 on different concentrations of B-a-Anth. as indicated in Table (51) and Figure (93) cleared that the maximum growth at the first three concentrations (100, 200 and 300 g/L) was reached after 24 hours. However, the higher concentration (400 and 500 g/L) B-a-Anth. need more time to reach the highest growth after six days incubation.

Extracellular protein reavealed that the maximum production was recorded at the third day followed by sharpe decrease till the fifth day as shown in Table (51) and Figure (94). This trend in protein production was similar with that of isolate MAM-26 and MAM-29 as previously mentioned.

211 Results and Discussion

Table (49): Growth and extracellular protein of strain MAM-26 on different concentrations of benzo-a-anthracene (B-a-Anth.). Conc. 200 300 400 500 100 (ug/L) Incubation (ug/L) (ug/L) (ug/L) (ug/L) period(days) Protein Protein Protein Protein Protein O.D. O.D. O.D. O.D. O.D. Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.390 60.8 0.406 62.0 0.403 59.8 0.389 60.8 0.399 64.0 1 0.415 47.3 0.399 52.0 0.389 38.5 0.390 56.2 0.419 72.3 2 0.410 73.0 0.407 61.5 0.407 70.7 0.387 53.8 0.393 72.3 3 0.357 116.9 0.383 123.0 0.522 131.9 0.363 104.2 0.397 117.6 4 0.378 85.4 0.382 77.3 0.383 87.7 0.363 74.6 0.382 85.0 5 0.338 50.0 0.381 38.5 0.365 49.2 0.297 32.7 0.376 31.5 6 0.387 53.0 0.375 30.0 0.379 44.0 0.362 30.0 0.376 30.0 7 0.354 49.9 0.365 33.0 o.356 42.0 0.351 29.9 0.370 27.8 14 0.380 44.0 0.369 32.0 0.372 40.0 0.353 27.0 0.367 26.9 21 0.331 40.0 0.347 30.0 0.351 39.6 0.326 25.0 0.376 25.0

212 Results and Discussion

100 (ug/l) 200 (ug/l) 300 (ug/l) 0.6 400 (ug/l) 500 (ug/l)

0.5

) 0.4 nm 0.3 600 . ( D .

O 0.2

0.1

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (89): Growth of strain MAM-26 on different concentrations of benzo-a-anthracene.

140 100 (ug/l) 200 (ug/l) 300 (ug/l)

120 400 (ug/l) 500 (ug/l)

100

80

60 Protein (ug/ml)Protein 40

20

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (90): Extracellular protein of strain MAM-26 on different concentrations of benzo-a-anthracene.

213 Results and Discussion

Table (50): Growth and extracellular protein of strain MAM-29 on different concentrations of benzo-a-Anthracene.

Conc. 100 200 300 400 500 Incubation (ug/L) (ug/L) (ug/L) (ug/L) (ug/L) period(days) Protein Protein Protein Protein Protein O.D. O.D. O.D. O.D. O.D. Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.190 74.6 0.171 72.0 0.167 76.0 0.163 74.6 0.146 75.4 1 0.188 41.2 0.197 47.5 0.186 44.2 0.175 42.0 0.167 43.0 2 0.172 53.8 0.177 57.7 0.174 58.5 0.163 60.0 0.152 41.5 3 0.171 83.5 0.168 96.5 0.170 101.0 0.171 102.0 0.144 83.8 4 0.164 67.3 0.168 76.5 0.169 82.3 0.151 79.6 0.141 63.0 5 0.157 15.7 0.157 26.9 0.154 29.2 0.150 31.9 0.140 28.5 6 0.157 18.9 0.164 26.0 0.148 29.9 0.147 30.0 0.134 30.0 7 0.140 20.0 0.148 25.0 0.142 26.0 0.141 32.0 0.125 29.9 14 0.134 22.0 0.149 24.0 0.141 23.0 0.134 30.0 0.115 25.0 21 0.134 18.5 0.143 22.0 0.137 24.0 0.126 26.0 0.125 23.9

214 Results and Discussion

100 (ug/l) 200 (ug/l) 300 (ug/l) 0.25 400 (ug/l) 500 (ug/l)

0.2 ) 0.15 nm 600 . ( D

. 0.1 O

0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (91): Growth of strain MAM-29 on different concentrations of benzo-a-Anthracene.

120 100 (ug/l) 200 (ug/l) 300 (ug/l)

100 400 (ug/l) 500 (ug/l)

80

60

Protein (ug/ml) 40

20

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (92): Extracellular protein of strain MAM-29 on different concentrations of benzo-a-anthracene.

215 Results and Discussion

Table (51): Growth and extracellular protein of strain MAM- 62 on different concentrations of benzo-a-anthracene (B-a- Anth).

Conc. 100 200 300 400 500 Incubation (ug/L) (ug/L) (ug/L) (ug/L) (ug/L) period(days) Protein Protein Protein Protein Protein O.D. O.D. O.D. O.D. O.D. Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.449 59.2 0.429 62.0 0.430 57.9 0.440 59.2 0.430 60.5 1 0.474 61.5 0.442 67.7 0.453 61.5 0.453 58.5 0.437 53.5 2 0.455 55.0 0.430 66.2 0.442 66.2 0.464 50.0 0.430 62.7 3 0.447 118.0 0.444 124.6 0.427 123.8 0.436 117.7 0.420 130.7 4 0.437 65.4 0.415 83.8 0.422 82.3 0.410 63.0 0.384 76.9 5 0.422 24.2 0.415 40.0 0.404 39.6 0.417 30.0 0.394 30.0 6 0.443 27.0 0.414 33.9 0.449 42.7 0.483 33.8 0.474 29.6 7 0.412 30.0 0.398 32.0 0.394 39.9 0.393 32.8 0.391 27.0 14 0.410 27.6 0.400 30.7 0.392 37.8 0.379 30.6 0.389 26.9 21 0.407 23.7 0.389 30.6 0.389 35.9 0.369 31.0 0.367 22.0

216 Results and Discussion

100 (ug/l) 200 (ug/l) 300 (ug/l) 0.6 400 (ug/l) 500 (ug/l) 0.5

) 0.4 nm

600 0.3 . ( D .

O 0.2

0.1

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (93): Growth of strain MAM-62 on different concentrations of benzo-a-anthracene.

140 100 (ug/l) 200 (ug/l) 300 (ug/l)

120 400 (ug/l) 500 (ug/l)

100

80

60 Protein (ug/ml) 40

20

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (94): Extracellular protein of strain MAM- 62 on different concentrations of benzo-a-anthracene.

217 Results and Discussion

Growth of isolate MAM-68 on different concentrations of B-a- Anth. indicated that the highest growth was recorded after 24 hours for concentrations (200, 300, 400 and 500 g/L) as shown in Table (52) and Figure (95). However, the lowest concentration (100 g/L) recorded the best growth at the second day.

Extracellular protein produced by MAM-68 has the same trend shown previously by the isolates MAM-26, MAM-29 and MAM-62. The maximum protein secreted was recorded at the third day followed by sharpe decrease till the fifth day as shown in Table (52) and Figure (96).

Profile of isolate E.cloacae MAM-4 on different concentrations of B-a-Anth. indicated that there were three peaks of growth at the first day , the sixth day and then after 14 days for the five concentrations as shown in Table (53) and Figure (97).

The same trend for extracellular protein secreted by E.cloacae MAM-4 on B-a-Anth. as previously mentioned in cases of MAM-26, MAM-29, MAM-62 and MAM-68. The maximum protein production was recorded at the third day followed by sharp decrease till fifth day as shown in Table (53) and Figure (98).

218 Results and Discussion

Table (52): Growth and extracellular protein of strain MAM- 68 on different concentrations of benzo-a-anthracene (B-a-Anth.).

Conc.

100 200 300 400 500

(ug/l) (ug/l) (ug/l) (ug/l) (ug/l) Incubation period(days) Protein Protein Protein Protein Protein O.D. O.D. O.D. O.D. O.D. Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.593 88.5 0.571 90.2 0.557 88.5 0.565 89.4 0.557 85.0 1 0.589 96.2 0.592 93.5 0.568 84.6 0.575 86.9 0.586 93.5 2 0.610 80.7 0.572 83.0 0.556 78.5 0.563 76.5 0.568 73.8 3 0.581 146.9 0.554 116.2 0.534 153.0 0.540 154.0 0.548 155.0 4 0.569 84.6 0.560 101.5 0.548 109.2 0.557 92.0 0.519 93.8 5 0.572 41.1 0.545 54.2 0.527 58.8 0.533 56.2 0.527 50.7 6 0.609 43.0 0.566 49.0 0.544 55.0 0.568 55.0 0.570 50.1 7 0.525 38.9 0.510 45.0 0.507 55.8 0.502 54.9 0.486 47.0 14 0.529 37.9 0.511 50.0 0.519 51.0 0.553 42.0 0.532 45.0 21 0.524 33.0 0.532 44.0 0.513 50.0 0.513 40.0 0.518 44.0

219 Results and Discussion

100 (ug/l) 200 (ug/l) 300 (ug/l) 0.7 400 (ug/l) 500 (ug/l) 0.6

0.5 )

nm 0.4 600

. ( 0.3 D . O 0.2

0.1

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (95): Growth of strain MAM- 68 on different concentrations of benzo-a-anthracene.

180 100 (ug/l) 200 (ug/l) 300 (ug/l) 160 400 (ug/l) 500 (ug/l) 140

120

100

80

Protein (ug/ml)Protein 60

40

20

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (96): Extracellular protein of strain MAM-68 on different concentrations of benzo-a-anthracene.

220 Results and Discussion

Table (53): Growth and extracellular protein of strain E.cloacae MAM- 4 on different concentrations of benzo-a- anthracene (B-a-Anth.). Conc. 100 200 300 400 500 Incubation (ug/l) (ug/l) (ug/l) (ug/l) (ug/l) period(days) Protein Protein Protein Protein Protein O.D. O.D. O.D. O.D. O.D. Zero time (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) 0.166 46.1 0.188 45.0 0.179 48.0 0.179 45.0 0.171 46.1 1 0.194 33.8 0.211 41.6 0.213 46.9 0.213 30.8 0.200 24.6 2 0.181 65.4 0.203 55.4 0.201 70.6 0.196 38.5 0.187 38.9 3 0.169 81.5 0.193 92.3 0.189 110.3 0.181 90.0 0.183 51.9 4 0.164 46.2 0.188 65.0 0.189 76.9 0.185 62.7 0.178 54.2 5 0.161 18.5 0.192 18.5 0.189 32.3 0.182 16.0 0.176 28.5 6 0.199 20.0 0.221 20.0 0.221 30.0 0.214 18.0 0.221 30.0 7 0.140 21.0 0.171 19.8 0.175 28.9 0.170 22.0 0.167 32.0 14 0.187 17.0 0.216 16.8 0.213 26.0 0.209 23.0 0.202 33.0 21 0.157 18.0 0.193 20.0 0.189 25.0 0.191 21.0 0.195 30.0

221 Results and Discussion

100 (ug/l) 200 (ug/l) 0.25 300 (ug/l) 400 (ug/l) 500 (ug/l)

0.2

) 0.15 nm 600 . ( D . 0.1 O

0.05

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (97): Growth of strain E.cloacae MAM-4 on different concentrations of benzo-a-anthracene.

180 100 (ug/l) 200 (ug/l) 300 (ug/l) 160 400 (ug/l) 500 (ug/l) 140

120

100

80

Protein (ug/ml)Protein 60

40

20

0 0 1 2 3 4 5 6 7 14 21 Incubation period (days)

Figure (98): Extracellular protein of strain E.cloacae MAM-4 on different concentrations of benzo-a-anthracene.

222 Results and Discussion

The count of isolate MAM-26 on the five different concentrations be more than the count of initial (9.8x107 CFU/ml) at the end of incubation period (21 days) as indicated in Table (54). Also, isolate E.cloacae MAM-4, four out of five concentrations tested, their counts exceed the initial count (1.7x107CFU/ml) at the end of incubation. The other three isolates (MAM-29, MAM-62 and MAM-68) non of the count exceed their initials (5.0x107; 58.0 x107 and 80x107 CFU/ml) respectively except the concentration of MAM-29 at 100 ug/l.

The most extensive degradation was apparent with the 2- and 3- ring PAH, with decreases of 97% and 82%, respectively. The higher molecular weight 3- and 4- ring PAH were degraded at slower rates, with reductions of 45% and 51% respectively. Six-ring PAH were degraded with least average reductions of 35% (Guerin, 1999).

Up to 70%, 86% and 84% of benzo(a) anthracene, benzo(a) pyrene, and dibenzo(a,h) anthracene, respectively, were removed in presence of fungi while the indigenous microorganism converted merely up to 29%, 26% and 43% of these compounds in 30 days. Low moelcular- mass PAH studied were easily degraded by soil microbes and only anthracene degradation was enhanced by the fungi well (Steffen et al., 2007).

The explantation may be that small PAHs serve as sole carbon source for certain bacteria and are intracellularly metabolized, whereas the larger PAHs enter the cell to a minor degree and cannot be utilized as growth substrate. The phenomenon is analogous for the inability of bacteria to degrade high molecular weight lignin and lignin model compounds (Hatakka, 2001).

223 Results and Discussion

Table (54): Count of the selected isolated strains on different concentrations of benzo-a-anthracene after 21 days incubation period.

Concentrations (ug/l)

Isolate Initial count 100 200 300 400 500 code (CFU/ml) Io

count logN count logN count logN count logN count logN count logN

MAM-26 9.8*107 7.99 11.0*107 8.04 12.0*107 8.07 10.4*107 8.01 10.0*107 8.00 12.0*107 8.07

MAM-29 5.0*107 7.69 6.3*107 7.79 1.5*107 7.17 2.5*107 7.39 4.5*107 7.65 4.4*107 7.64

MAM-62 58.0*107 8.76 6.3*107 7.79 9.0*107 7.95 14.8*107 8.17 12.0*107 8.07 21.0*107 8.32

MAM-68 80*107 8.90 9.0*107 7.95 16.5*107 8.21 8.0*107 7.90 8.0*107 7.90 1.2*107 7.07

E.cloacae 1.7*107 7.23 1.3*107 7.11 2.7*107 7.43 1.8*107 7.25 2.8*107 7.44 5.8*107 7.76 MAM-4

224 Results and Discussion

White rot fungi also degraded Benzo(a)pyrene (Barclay et al., 1995 and Kotterman et al., 1998).

A 16S rRNA gene sequence analysis and biochemical tests identified strain BPC1 as a member of the genus Rhodanobacter whose type strain R. lindaniclasticus RP 5557. This strain degraded Benzo(a)pyrene (Kamaly et al., 2002).

Degradation percentage of B-a-Anth. by different isolates had been determined as shown in Table (55) and Figure (100) the results of degradation as determined by HPLC indicated that the best B-a-Anth. degrader was isolate MAM-26 followed by MAM-29. Isolate MAM-26 degraded 60% of 100 g/L B-a-Anth and 64% of 500 g/L B-a-Anth. However, isolate MAM-29 degraded 63% of 100 g/L and 62% of 500 g/L B-a-Anth. The third best B-a-Anth. degrader was isolate MAM-62. This isolate degraded 38% of 100 g/L and 39% of 500 g/L B-a-Anth.

Although, MAM-62 was the best degrader of Naph., Phen., Anth. and the second degrader for pyrene it was the third degrader for B-a-Anth.

225 Results and Discussion

Table (55): Degradation percentage of benzo-a-anthracene after 21 days by HPLC.

Degradation % Isolate code 100 200 300 400 500 (ug/l) (ug/l) (ug/l) (ug/l) (ug/l)

MAM-26 60% 57% 42% 55% 64%

MAM-29 63% 53% 52% 52% 62%

MAM-62 38% 58% 36% 18% 39%

MAM-68 17% 26% 28% 9% 32%

E. cloacae MAM- 7% 11% 15% 6% 18% 4

MAM-26 MAM-29 MAM-62 MAM-68 E. cloaceae MAM-4

70%

60%

50%

40%

30% Degradation % Degradation 20%

10%

0% 100 200 300 400 500

Concentration (ug/L)

Figure (99): Degradation percentage of benzo-a-anthracene after 21 days by HPLC.

226 Results and Discussion

3.9. Identification of the most potent PAHs degrading bacterial strains.

From the the previous results, we select isolate MAM-29 and MAM-62 as Gram negative and Gram positive isolates that represent the best PAHs degrading bateria. The Gram positive MAM-62 was characterized as Gram positive, spore-forming Bacillus, creamy, smooth large, irregular margin, flat when grown on L.B agar plates. However, the Gram negative isolate MAM-29 was characterized as short rods Gram negative, yellowish to creamy, medium, smooth, flat when growth on L.B agar plates too.

DNA extracted from isolates MAM-29 and MAM-62 were amplified by PCR. The products of PCR were electrophoresed by agarose gel electrophorsis as indicated in Figure (100). DNA were about 1500 kbp. The nucleotide sequences of the 16S rRNA from the isolated strain MAM- 29 was determined comprising 944 and that of isolated strain MAM-62 was 1154 nucleotides and deposited in the NCBI gene bankit sequences databases under accession numbers (JN 038055) and (JN 038054) as shown in Figures (101-104) respectively.

227 Results and Discussion

Figure (100): Agarose gel of DNA of isolated strains MAM-29 and MAM-62 polycyclic aromatic hydrocarbon degrading bacteria

228 Results and Discussion

1 tatcggatta ctgggcgtaa gcgtgcgcag gcggttcgga aagaaagatg tgaaatccca 61 gagcttaact ttggaactgc atttttaact accgggctag agtgtgtcag agggaggtgg 121 aattccgcgt gtagcagtga aatgcgtaga tatgcggagg aacaccgatg gcgaaggcag 181 cctcctggga taacactgac gctcatgcac gaaagcgtgg ggagcaaaca ggattagata 241 ccctggtagt ccacgcccta aacgatgtca actagctgtt ggggccttcg ggccttggta 301 gcgcagctaa cgcgtgaagt tgaccgcctg gggagtacgg tcgcaagatt aaaactcaaa 361 ggaattgacg gggacccgca caagcggtgg atgatgtgga ttaattcgat gcaacgcgaa 421 aaaccttacc tacccttgac atgtctggaa tgccgaagag atttggcagt gctcgcaaga 481 gaaccggaac acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa 541 gtcccgcaac gagcgcaacc cttgtcatta gttgctacga aagggcactc taatgagact 601 gccggtgaca aaccggagga agggtggggg atgacgtcaa gtcctcatgg cccttatggg 661 gtagggcttc acacgtcata caatggtcgg gacagagggt cgccaacccg cgagggggag 721 ctaatcccag aaacccgatc gtagtccgga tcgcagtctg caactcgact gcgtgaagtc 781 ggaatcgcta gtaatcgcgg atcagcatgt cgcggtgaat acgttcccgg gtcttgtaca 841 catcgcccgt caaccatggg gagagttggg ttttactcag aagtagttag cctaaccgca 901 aaggaggtgc gattaccacc gtagatcatg actggggtga agt Figure (101): DNA sequencing of isolate MAM-29.

Figure (102): Phylogenetic tree constructed to isolated strain MAM-29.

229 Results and Discussion

1 tccattgtag cacgtgtgta gcccaggtca taaggggcat gatgatttga cgtcatcccc 61 accttcctcc ggtttgtcac cggcagtcac cttagagtgc ccaacttaat gatggcaact 121 aagatcaagg gttgcgctcg ttgcgggact taacccaaca tctcacgaca cgagctgacg 181 acaaccatgc accacctgtc actctgctcc cgaaggagaa gccctatctc tagggttttc 241 agaggatgtc aagacctggt aaggttcttc gcgttgcttc gaattaaacc acatgctcca 301 ccgcttgtgc gggcccccgt caattccttt gagtttcagc cttgcggccg tactccccag 361 gcggagtgct taatgcgtta acttcagcac taaagggcgg aaaccctcta acacttagca 421 ctcatcgttt acggcgtgga ctaccagggt atctaatcct gtttgctccc cacgctttcg 481 cgcctcagtg tcagttacag accagaaagt cgccttcgcc actggtgttc ctccatatct 541 ctacgcattt caccgctaca catggaattc cactttcctc ttctgcactc aagtctccca 601 gtttccaatg accctccacg gttgagccgt gggctttcac atcagactta agaaaccacc 661 tgcgcgcgct ttacgcccaa taattccgga taacgcttgc cacctacgta ttaccgcggc 721 tgctggcacg tagttagccg tggctttctg gttaggtacc gtcaaggtgc cagcttattc 781 aactagcact tgttcttccc taacaacaga gttttacgac ccgaaagcct tcatcactca 841 cgcggcgttg ctccgtcaga ctttcgtcca ttgcggaaga ttccctactg ctgcctcccg 901 taggagtctg ggccgtgtct cagtcccagt gtggccgatc accctctcag gtcggctacg 961 catcgttgcc ttggtgagcc gttacctcac caactagcta atgcgacgcg ggtccatcca 1021 taagtgacag ccgaagccgc ctttcaattt cgaaccatgc ggttcaaaat gttatccggt 1081 attagccccg gtttcccgga gttatcccag tcttatgggc aggttaccca cgtgttactc 1141 acccgtccgc cgc

Figure (103): DNA sequencing of isolate MAM-62.

Figure (104): Phylogenetic tree constructed to isolated strain MAM-62.

230 Results and Discussion

A phylogenetic tree constructed on the obtained 16S-rRNA coding gene sequences of isolates MAM-29 and 62 and the nearst relatives were shown in Fig (102 and 104). The 16S-rRNA of isolate MAM-29 showed similarity of 100% to Achromobacter sp. with accession No JN 038055.1 and Achromobacter xylosoxidans strain R8-558 with accession No. JQ659958.1. So isolate MAM-29 was identified as Achromobacter xylosoxidans with accession No. JN 038055.

It showed also 81% similarity with A. sp. with accession No. HM 071054.1

However, the 16S-rRNA of isolate MAM-62 showed a similarity of 99% to Bacillus amyloliquefaciens with accession No. FJ009402, Bacillus sonorensis with accession No. AJ 586363 and Bacillus macroides with accession No. DQ 350821. Also it showed 72% similarity to B. cereus Jx 195185, B. subtilis FJ 435215 and B. thuringiensis JQ 342840.

So, the isolate MAM-62 was identified as Bacillus amyloliquefaciens with accession No. JN038054. Another investigators also identified PAHs degrading bacterial strains by 16S r-RNA as the following.

Representative strains of CB-BT, identified on the basis of 1200 nucleotides sequence homologies with entries in GenBank-EMBL databases, belong to: Achromobacter xylosoxidans (100%), Methylobacterium sp (99%), Alcaligenes sp. (99%), Rhizobium galegae (99%), R.aetherovorans (100%), Aquamicrobium defluvium (100%) (Andreoni et al., 2004).

The high percentage of Bacillus strains characterized in our work (66.6%) could be related with the property of these microorganisms to colonize environments contaminated with hydrocarbons (Zhuang et al., 2002). 231 Results and Discussion

Toledo et al. (2006) identified strain GT2B by sequencing the 16s rDNA gene with a continuous streach of > 1400 nucleotides. The results revealed that most strains were belong to genera of Bacillus.

The isolated Gram negative bacterium isolated from contaminated soil was identified based on 16S rDNA. The strain GT2B was closely related to species in genus Sphingomonas with 99.15%. Similarity to S. chungbukensis (AT151392) (Tao et al., 2007).

Weid et al. (2007) isolatd and identified strain P4 by its partial 16S rRNA gene sequence. The results showed that the position of strain P4 closely associated with spices of the genus Dietzia with similarities of 99.4%, 98.3%, 98.3%, 99.4 and 99.8% to D. maris, D. Kunjamensis, d. psychralcaliphila, D. natronolimnaea and D. cinnamea respectively.

Pathak et al. (2009) found that DNA sequencing and BLAST analysis of complete 16S rRNA gene sequence of strain HOB1 showed maximum sequence indentity 97% with .

Obuekwe et al. (2009) isolated and identified the prominent crude oil-utilizers by 16S rDNA sequence analysis. Members of Bacillus spp. constitute the dominant group. Bacillus Licheniformis (54%) and Bacillus cereus (15%) were the doment species of Bacillus isolates.

Zhang et al. (2009) identified the isolated strain JY11 by sequencing 16S rDNA-based phylogentic analysis which demonstrated that the strain belonged to the genus Janibacter. The similarity between strain JY11 with J.anaphelis (99.93%), J.terrae (98.48%), J. marinus (98.38%), J.limosus 98.34%, J. melonis (98.20%) and J.corallicola (97.79%).

Identified, based on 16S rDNA, 23 clones of identifying bacteria (19 clones out of 23 clones) was mainly composed of -proteobacteria and

232 Results and Discussion

4 clone belong to Actinobacter having the ability to degrade naphthalene (Lu et al., 2011).

3.10. Effect of gamma radiation on the viability of Bacillus amyloliquefaciens.

In this study, Bacillus amyloliquefaciens accession No. (JN038054) was chosen to be exposed to different doses of gamma radiation as a best degrader for PAHs degrading bacteria. The dose response curve of γ-radiation, revealed that, as the dose increased the count of Bacillus amyloliquefacines decreased as indicated in Table (56) and Fig. (105).

The results also revealed that a rapid decrease had been shown till 4.0 kGy. Exposure to 4.0 kGy reduced the viable count by 5.25 Log cycles. Further increasing the doses of γ-radiation up to 15.0 kGy decreased the viable count with decreasing rates. Exposure of B.amyloliquefaciens (JN038054) to more γ-radiation (4-15 kGy) reduced the viable count by 2.0 log cycles as in Fig. (105). This phenomena may be explained on the basis that rapid decrease at the first part of the curve was for the death of vegetative cells of B.amyloliquefacies (JN038054), while the decreased decline in count at the higher doses of exposure may be rely to the resistance spores of B.amyloliquefaciens (JN038054) like other strains of bacilli which show a high resistant to gamma radiation. These results were in a good agreement with that reported by Abo-State (1991, 1996, and 2004), Abo-State and Khalil (2001); and Abo-State et al. (2005).

233 Results and Discussion

Table (56): Effect of gamma irradiation on the viability of B. amyloliquefaciens.

Dose (kGy) Count CFU/ml Log N Control 18.0x109 8.25 1 100.0x104 6.00 2 2.0x104 4.30 4 0.1 x104 3.00 6 0.03 x104 2.47 8 0.018 x104 2.25 10 0.01 x104 2.200 12 0.002 x104 1.30 15 0.001 x104 1.00

Figure (105): Effect of gamma-radiation doses on the viable count of isolated strain B. amyloliquefaciens

234 Results and Discussion

3.11 Selection of the hyper PAHs degrading bacterial mutant.

Colonies resulted from exposure of the parent strain B.amyloliquefaciens to different doses of gamma irradiation, that having morphological changes on L.B agar medium were picked up as separated single colonies. Twenty four colonies were selected with that of the parent strain (wild type) to be grown on BSM supplemented with PAH compounds (1000 mg/L Naph.; 750 mg/L Phen.; 75 mg/L Anth.; 300 g/L Pyr. And 300 g/L B-a-Anth.) for 1,2 and 7 days. The best mutant (colony) grown on PAHs was mutant No. “4” as indicated from Table (57) and (58). The results revealed that mutant No. ”4” had not increased growth on Naph. than the parent strain, but it showed great increase in growth on Phen., Anth., Pyr. and B-a-Anth. The increases were 2.0, 1.5, 0.97; 2.5, 1.2, 2.5; 9.5, 12.5, 7.5; 7.5, 12.5 and 5.0 folds, after 1, 2 and 7 days respectively (Table 58). These results indicated that mutant No. “4” showed superior growth on the four PAH compounds especially on Pyrene and Benzo-a-anthracene when compared with parent strain.

235 Results and Discussion

Table (57): Growth of the parent strain of Bacillus amyloliquefaciens and its different selected mutants isolated exposed to different doses of gamma radiation on different PAHs.

Compounds Naphthalene (1000 mg/L) Phenanthracene (750 mg/L) Anthracene (75 mg/L) Pyrene (300 ug/L) B-a-anthracene (300 ug/L) Dose First Second Seventh First Second Seventh First Second Seventh First Second Seventh First Second Seventh (KGy) Mutant no I I I I I o day day day o day day day o day day day o day day day o day day day Wild type- Zero 0.05 0.072 0.056 0.037 0.053 0.097 0.085 0.136 0.047 0.098 0.141 0.098 0.047 0.08 0.056 0.037 0.036 0.067 0.056 0.052 parent strain.

8 1 0.023 0.06 0.02 0.022 0.03 0.12 0.055 0.139 0.028 0.033 0.039 0.058 0.028 0.036 0.021 0.034 0.045 0.033 0.012 0.017

8 2 0.12 0.129 0.103 0.082 0.13 0.159 0.108 0.135 0.126 0.152 0.115 0.114 0.109 0.111 0.085 0.112 0.117 0.125 0.104 0.1

8 3 0.099 0.088 0.06 0.072 0.13 0.176 0.137 0.156 0.121 0.123 0.131 0.115 0.121 0.13 0.092 0.078 0.107 0.121 0.085 0.102

1 4 0.016 0.016 0.01 0.01 0.046 0.092 0.07 0.045 0.012 0.03 0.015 0.03 0.002 0.019 0.025 0.015 0.002 0.015 0.025 0.01

1 5 0.101 0.16 0.117 0.127 0.12 0.198 0.1 0.096 0.156 0.194 0.121 0.135 0.125 0.165 0.162 0.116 0.1 0.185 0.13 0.163

2 6 0.028 0.062 0.027 0.024 0.063 0.09 0.07 0.08 0.076 0.086 0.039 0.086 0.108 0.052 0.035 0.028 0.1 0.07 0.03 0.027

4 7 0.106 0.104 0.08 0.07 0.108 0.104 0.113 0.185 0.127 0.1 0.07 0.045 0.112 0.109 0.077 0.067 0.122 0.09 0.066 0.1

4 8 0.044 0.042 0.019 0.027 0.096 0.162 0.094 0.134 0.06 0.091 0.047 0.045 0.036 0.039 0.034 0.024 0.046 0.053 0.022 0.036

6 9 0.05 0.044 0.069 0.035 0.112 0.137 0.124 0.19 0.045 0.059 0.035 0.061 0.047 0.059 0.01 0.026 0.044 0.059 0.034 0.027

6 10 0.046 0.06 0.029 0.035 0.081 0.105 0.085 0.073 0.055 0.08 0.065 0.08 0.045 0.047 0.036 0.038 0.05 0.053 0.042 0.037

236 Results and Discussion

Table (57): Continue

Compound Naphthalene (1000 mg/L) Phenanthracene (750 mg/L) Anthracene (75 mg/L) Pyrene (300 ug/L) B-a-anthracene (300 ug/L) Dose (KGy) First Second Seventh First Second Seventh First Second Seventh First Second Seventh First Second Seventh Mutant no I I I I I o day day day o day day day o day day day o day day day o day day day

6 11 0.031 0.038 0.061 0.025 0.048 0.08 0.117 0.08 0.039 0.083 0.1 0.076 0.038 0.054 0.04 0.027 0.036 0.051 0.042 0.01

6 12 0.13 0.163 0.14 0.13 0.152 0.191 0.225 0.24 0.136 0.163 0.15 0.113 0.133 0.158 0.136 0.12 0.124 0.165 0.137 0.128

6 13 0.105 0.123 0.13 0.112 0.11 0.16 0.158 0.143 0.117 0.142 0.124 0.131 0.102 0.13 0.12 0.104 0.09 0.107 0.104 0.094

6 14 0.028 0.03 0.045 0.035 0.036 0.094 0.124 0.164 0.031 0.054 0.056 0.032 0.029 0.047 0.019 0.001 0.024 0.03 0.026 0.015

10 15 0.026 0.026 0.035 0.02 0.023 0.071 0.12 0.101 0.019 0.075 0.095 0.078 0.014 0.042 0.035 0.02 0.015 0.035 0.036 0.021

10 17 0.045 0.047 0.084 0.05 0.047 0.09 0.152 0.181 0.054 0.073 0.096 0.063 0.052 0.089 0.095 0.055 0.043 0.04 0.07 0.025

10 18 0.032 0.047 0.025 0.04 0.027 0.065 0.075 0.1 0.018 0.067 0.076 0.046 0.025 0.048 0.034 0.017 0.029 0.041 0.022 0.02

12 21 0.063 0.046 0.021 0.03 0.067 0.07 0.076 0.067 0.051 0.032 0.022 0.051 0.06 0.045 0.026 0.05 0.063 0.043 0.02 0.015

15 22 0.011 0.037 0.03 0.02 0.014 0.078 0.138 0.143 0.014 0.037 0.017 0.026 0.005 0.034 0.011 0.001 0.01 0.032 0.011 0.02

15 23 0.036 0.043 0.029 0.016 0.072 0.121 0.143 0.155 0.063 0.064 0.082 0.07 0.045 0.017 0.032 0.01 0.036 0.026 0.044 0.01

12 24 0.15 0.12 0.124 0.077 0.152 0.138 0.151 0.093 0.127 0.11 0.148 0.107 0.136 0.111 0.126 0.104 0.133 0.128 0.121 0.089

Io=O.D. at zero time 1,2,7=days of incubation

237 Results and Discussion

Table (58): The increase in growth of the selected mutants (I) on different PAHs after different incubation periods compared to the parent strain(Io)

Mutant Naphthalene (I/I0) phenanthrene(I/I0) anthracene(I/I0) pyrene(I/I0) B-a-anthracene(I/I0) no 1 2 7 1 2 7 1 2 7 1 2 7 1 2 7 Parent 1.440 1.120 0.740 1.830 1.603 2.566 2.085 3.000 2.085 1.702 1.191 0.787 1.861 1.555 1.444 strain 1 2.608 0.086 0.095 4.000 1.833 4.633 1.178 0.714 2.071 1.285 0.750 1.214 0.733 0.266 0.377 2 1.075 0.858 0.683 1.223 0.830 1.038 1.206 0.912 0.904 1.018 0.779 1.027 1.068 0.888 0.854 3 0.888 0.606 0.727 1.353 1.053 1.200 1.016 1.082 0.950 1.074 0.760 0.644 1.130 0.794 0.953 4 1.000 1.000 1.000 2.000 1.521 0.978 2.500 1.250 2.500 9.500 12.50 7.500 7.500 12.500 5.000 5 1.584 1.158 1.257 1.650 0.833 0.800 1.243 0.775 0.865 1.320 1.296 0.928 1.850 1.300 1.630 6 2.214 0.964 0.857 1.428 1.111 1.269 1.131 0.513 1.131 0.481 0.324 0.259 0.700 0.300 0.270 7 0.098 0.754 0.660 0.962 1.046 1.712 0.787 0.551 0.354 0.973 0.687 0.598 0.737 0.540 0.819 8 0.954 0.431 0.613 0.168 0.979 1.395 1.516 0.783 0.750 1.083 0.944 0.666 1.152 0.478 0.782 9 0.880 1.380 0.700 1.223 1.107 1.696 1.311 0.777 1.355 1.255 0.212 0.553 1.340 0.772 0.613 10 1.304 0.630 0.760 1.296 1.049 0.901 1.454 1.181 1.454 1.044 0.800 0.844 1.060 0.084 0.740 11 1.225 1.967 0.806 1.666 2.437 1.666 2.128 2.564 1.948 1.421 1.052 0.710 1.416 1.166 0.277 12 1.253 1.076 1.000 1.256 1.480 1.578 1.198 1.102 0.830 1.187 1.022 0.902 1.330 1.104 1.045 13 1.171 1.238 1.066 1.454 1.436 1.578 1.213 1.059 1.119 1.274 1.176 1.019 1.188 1.155 1.044 14 1.071 1.607 1.250 2.611 3.444 4.555 1.741 1.806 1.032 1.620 0.655 0.034 1.250 1.083 0.625 15 1.000 1.346 0.769 3.086 5.217 4.39 3.947 5.000 4.105 3.000 2.500 1.428 2.333 2.500 0.714 17 1.044 1.866 1.111 1.914 3.234 3.851 1.370 1.777 1.166 1.711 1.826 1.057 0.930 1.627 0.581 18 1.468 0.781 1.250 2.407 2.777 3.703 3.722 4.222 2.555 1.920 1.360 0.680 1.413 0.758 0.689 21 0.730 0.333 0.476 0.957 1.134 1.000 0.627 0.431 1.000 0.750 0.433 0.833 0.682 0.317 0.238 22 3.363 2.727 1.818 5.571 9.857 10.214 2.642 1.214 1.857 6.800 2.200 0.200 3.200 1.100 1.000 23 1.194 0.805 0.444 1.680 1.986 2.152 1.015 1.301 1.111 0.377 0.711 0.222 0.722 1.222 0.277 24 0.800 0.826 0.513 0.907 0.993 0.611 0.866 1.165 0.842 0.816 0.926 0.764 0.962 0.909 0.669

238 Results and Discussion

3.12. Pathway of B. amyloliquefaciens for degradation of PAH compounds.

Large number of studies investigated the pathway of the Gram negative bacteria especially Pseudomonas spp. Also investigated the pathway of the Gram-positive bacteria, Rhodococcus, but a little or nearly non of the studies investigated the pathway of Bacillus spp. to degrade PAHs. So, the parent strain and mutant No. “4” were grown in large quantity on different PAHs for GC-MS analysis after 24 hours incubation. This incubation period had been selected to determine different metabolites formed in degradation of PAHs.

The results of GC/MS analysis as indicated in Table (59) and Fig. (106) revealed that B. amyloliquefaciens MAM-62 degraded naphthalene to give 23 intermediate compounds, meanwhile the mutant of B. amyloliquefaciens MAM-62(4) degraded naphthalene to give 9 intermediate compounds as indicated in Fig. (107). Six intermediates compounds were found in both parent B. amyloliquefaciens MAM-62 and its mutant B. amyloliquefaciens MAM-62 (4). These compounds were Heptanoic acid, Hexanoic acid 2-ethyl, Nonanoic acid, 2-methyl indanone, Indol-5-aldhyde and Hexadecanonic acid.

It is clear that B. amyloliquefaciens MAM-62 undergo oxidation followed by ring fission to give benzene acetic acid and benzaldhyde with more oxidation and ring fission, about 10 intermediate compounds formed which undergo a kind of in aliphatic chain mannar. The above results have been compared with results of other investigators.

239 Results and Discussion

Table (59): Intermediates determined by GC-MS analysis of Naphthalene degradation by B. amyloliquefaciens and its mutant MAM-62(4) after 24 hours incubation.

R.T MAM-62 Formula MAM-62(4) Formula

11.446 C7H6O - -

12.169 - - 6-methyl-5-hepten- C8H14O 2-one

12.208 Hexanoic acid C6H12O2 - -

13.475 Octa-1,5dien-3-ol C8H14O - -

15.024 Heptanoic acid C7H14O2 Heptanoic acid C7H14O2

16.442 Hexanoic acid -2- C8H16O2 Hexanoic acid-2- C8H16O2 ethyl ethyl

18.761 Napthalene C10H8 Napthalene C10H8

18.820 Benzo(b)thiophene C10H6S - -

19.608 - - Propanamide, N- C9H19NO (1,1- dimethylethyl)2,2- dimethyl

20.225 Benzene acetic acid C8H8O2

20.747 Nonanoic acid C9H18O Nonanoic acid C9H18O2

21.804 2-methylindanone C10H10O 2-methyllindanone C10H10O

23.308 n-Decanoic acid C10H20O2 - -

25.334 1-H-inden-1-ol-2,3 C11H14O - - dihydro-3,3- dimethyl

25.655 acetic C11H20O2 - - acid 2-hexyl

25.657 - - Acenaphthalene C12H8

25.726 Undecanoic acid C11H22O2 - -

26.817 Phenol2,4-bis (1,1 C14H22O - - dimethylethyl)

28.099 Dodecanamide N, N C16H33NO - - bis (2hydroxyethyl)

240 Results and Discussion

Table (59): Continue

R.T MAM-62 Formula MAM-62(4) Formula

29.921 Phophoric acid tri C12H27O4P - - butylester

30.971 2(3H)-benzothia- C7H5NOS - - zolone

32.500 - - Benzoic acid peta- C7HCl5O2 chloro

32.503 Tetradecanoic acid C14H28O2 - -

33.877 Indol-5-aldhyde C9H7NO Inol-5-aldhyde C9H7No 36.576 Hexadecanoic acid C16H32O2 Hexadecanoic C16H32O2 acid 39.776 2-Azido-2,4,4,6,6- C12H25N3 - - pentamethyl 40.513 Hexadecanoic acid C20H40O2 - - butylester 43.837 Octadecanoic acid C22H44O2 - - butylester

241 Results and Discussion

S 1) ring fission 2) sulfurization

Benzo(b) thiophene 1) Doxidation 1) Oxidation 2) Ring fission 2) Ring fission O 3) Amination O OH Naphthelene

N 1) Ring fission H Benzene acetic acid 2) Amination and sulfurization Indol-5-aldhyde OH Oxidation

O H N 1H-Inden-1-ol O 1) Reduction 2,3 dihydro-33-di methyl 2) Substitution S

Benzaldhyde 2(3H)-Benzothiazolone

O 1) Oxidation 10 9 8 7 6 5 4 3 2 1 2) Ring fission O 3) Polymerization Octadecanoic acid butyl

O (I) Hexadecanoic acid butyl ester O a) Hexanoic acid OH 9 8 7 6 5 4 3 2 1 O O

b) Hexanoic acid 2-ethyl OH 1 2 3 4 5 O O OH

c) Hepatonic acid O OH F) Undecanoic acid OH 1 2 3 4 5 6 O

OH G) Tetra decanoic acid

1 2 3 4 5 6 7 O

OH H) n-hexadecanoic acid 4 3 2 1 OH d) Nonanoic acid O O 4 3 2 1 OH e) n-Decanoic acid Fig. (106): Proposed pathway for the degradation of naphthalene by B. amyloliquefaciens MAM-62.

242 Results and Discussion

O

5-heptanen 2-one 6 methyl 1) Oxidation Heterocyclic 2) Ring fission formation Heptanoic acid - 2-ethyl- Acenaphthene

O OH Naphthalene HO O Polymerization 1) Oxidation 1) Oxidation 2) Ring fission 2) Ring fission Cl Cl Hepatonic acid 3) Amination 3) Chlorination O Cl Cl Cl OH O

OH H Benzoic acid Pentachloro Nonanoic acid O N

Indol-5-aldhyde

Fig. (107): Proposed pathway for the degradation of naphthalene by the mutant of B. amyloliquefaciens MAM-62(4).

243 Results and Discussion

The proposed pathway for Naphthalene by B. amyloliquefaciens contains a set of metabolites not found between the other pathways mentioned before, like; Benzo(b) thiophene, Indol-5- and thiazolone ester and for it's mutant; Acenaphthene , Benzoic acid pentachloro and -5 aldehyde

The proposed pathway for Pseudomonas putida by (Reshetilov et al., 1997), cleared that its metabolites were; salicylate catechol; catechol and muconic acid.

However , the pathway which indicated by Gopishethy et al. (2007) revealed that S. griseus had the intermediates: hydroxy tetralone, methyl Naphthoquinone, methyl hydroxy tetralone, Naphthol and tetralone. The results of the present study revealed that the proposed pathway is a new pathway for naphthalene by B. amyloliquefaciens.

The results of degradation of phenanthrene GC/MS as indicated in Table (60) and Fig. (108, 109) revealed that B. amyloliquefaciens MAM- 62 produced 5 intermediate compounds while its mutant B. amyloliquefaciens MAM-62(4) produced 7 intermediate compounds. The only compound which found in both parent strain (B. amyloliquefaciens MAM-62) and its mutant (B. amyloliquefaciens MAM-62(4)) was Hexanoic acid.

Phenanthrene undergo oxidation to give 4(1H)-phenathrenone 2,3 dihydro with ring fission and more oxidation transformed to dimethylphathalate by B. amyloliquefaciens MAM-62. However, oxidation of phenanthrene by mutant B. amyloliquefaciens MAM-62(4) produced 1,2,3,4 teterhydro-phenanthrene -4-ol, phenanthrene-9-methoxy and 9-phenanthrenol. Further oxidation and ring fission produced hexanoic acid and Hexanoic acid 2,ethyl (aliphatic acids).

244 Results and Discussion

Table (60): Intermediates determined by GC-MS analysis of Phenanthrene degradation by B. amyloliquefaciens MAM-62 and its mutant MAM-62(4) after 24 hours incubation.

R.T MAM-62 Formula MAM-62(4) Formula

12.082 Hexanoic acid C6H12O2 Hexanoic acid C6H12O2

16.200 Hexanoic acid 2- C8H16O2 - - ethyl-

16.448 - - N-methyl-3-peperidine C7H14N2O carboxamide

19.597 Benzothiazole C7H5NS - -

25.488 Dimethylphathalate C10H10O4 - -

26.879 - - ,2,4-bis(1,1- C14H22O dimethylethyl-)

33.675 Phenanthrene C14H10 Phenanthrene C14H10

36.712 4(1H)- C14H12O - - Phenanthrenone 2,3-dihydro

38.305 - - 1,2,3,4- C14H14O tetrahydrophenanthrene- 4-ol

39.595 - - Phenanthrene,9- C15H12O methoxy-

40.386 - - Phenethrol C14H10O

40.575 - - 1-H-Phenanthro[9,10-C] C15H10N2 pyrazole

245 Results and Discussion

Oxidation Phenanthrene

O

4 (1H)-Phenanthrenone 2,3 dihydro

1) Ring fission 2) More oxidation

O 1) Reduction O 2) Amination and sulpherization N O S Benzothiazole Dimethyl phathalate

O

a) Hexanoic acid 1) Ring fission OH 2) Polymerization b) Hexanoic acid 2-ethyl O OH Fig. (108): Proposed pathway for the degradation of Phenanthrene by B. amyloliquefaciens MAM-62.

246 Results and Discussion

O 1) Oxidation 2) Ring fission N 3) Methylation and amination Heterocyclic addition N H NH2 Phenanthrene N 1-H-Phenanthro (9,10c) pyrazole

N. methyl 3, Oxidation peperidine carboxamide Oxidation 1) Oxidation 2) Methylation

OH

OH 9-Phenanthrenol

1, 2, 3, 4 Tetrahydro Phenanthrene 4-ol 1) Oxidation 2) Ring fission 3) Substitution O Phenanthrene 9- methoxy 1) Oxidation 2) Ring fission OH

a) Hexanoic acid

O Phenol 2,4-bis OH (1,1dimethyl ethyl)

b) Hexanoic acid 2,ethyl

O OH Fig. (109): Proposed pathway for the degradation of Phenanthrene by the mutant of B. amyloliquefaciens MAM-62 (4).

247 Results and Discussion

The proposed pathway for phenanthrene by P. amyloliquefaciens contains a set of metabolites not present in the other proposed pathways before, like (in case of wild type); phenanthrenone dihydro, dimethyl phthalate benzothiazole and for it's mutant the metabolites are, phenanthro pyrazole, N-methyl peperidine carboxamide. These metabolites are not found in the other proposed pathways, (Tao et al., 2007) by Sphingomonas sp. in which it's metabolites are naphthoic acid, naphthol, salicylic acid, catechol and in other proposed pathway by (Haritash and Kaushik, 2009) for aerobic bacteria, like; dihydrodiol, catechol, muconic acid and other metabolites reported by (Luan et al., 2006) for bacterial consortium like dihydroxy phenanthrene, benzoic acid benzoyl methyl ester and phthalatic acid and for (Benzalel et al., 1996) by Pleurotus ostreatus are phenanthrene oxide, phenanthrene dihydrodiol, quinone and diphenoic acid.

The results of GC/MS analysis of anthracene degradation by B. amyloliquficans as indicated in Table (61) and Fig. (110) revealed that, the parent strain B. amyloliquefaciens MAM-62 produced 6 intermediate compounds, while its mutant strain MAM-62(4) produced 4 intermediates. The cornstone in anthracene degradation was napthalane which produced in case of both the parent strain and its mutant. With more oxidation and ring fission both the parent strain and mutant intermediates undergo polymerization to give n-Hexadecanoic acid or tridecanol respectively.

248 Results and Discussion

Table (61): Intermediates determined by GC-MS analysis of anthracene degradation by B. amyloliquefaciens MAM-62 and its mutant MAM-62(4) after 24 hours incubation.

R.T MAM-62 Formula MAM-62(4) Formula

14.907 Hexachloroethane C2Cl6 - -

18.339 Napthalene C10H8 Naphthalene C10H8

19.492 - - 3-Hexanone-4-methyl C7H14O

29.515 - - Octadecanoic acid C18H34O2

30.690 - - Tridecanal C13H26O

33.670 Hexachlorobenzene C6Cl6 - -

33.670 Anthracene C14H10 Anthracene C14H10

34.723 Thioxanthene C13H10S - -

36.505 7,9-Ditert-butyl-1- C17H24O3 - - oxaspiro(4,5)deca- 6,9--2,8-dione

36.599 Hexadecanoic acid C16H30O2 - -

249 Results and Discussion

O

7,9-Di-tetra-butyl-1-oxospiro (4,5) deca-6,9 diene 2,8 dione O

1) Oxidation O 2) Ring fission Parent strain MAM-62 Mutant MAM-62 (4) 3) Side chain substitution 1) Oxidation 2) Riung fission

1) Oxidation Naphthalene Anthracene 2) Ring fission

Naphtalene 1) Oxidation 1) Oxidation 2) Ring fission 2) Ring fission 3) Chloronation 3) Chloronation 1) Reduction by sulphenization 1) Oxidation O Cl 2) Ring fission 3) Polymerization Cl Cl

3- Hexanone Cl Cl 4- Methyl S Cl Thioxanthene B enzene hexachloro O

b) 6-chloro 1) Oxidation OH Cl Cl 2) Ring fission Cl Cl 3) Polymerization Cl Cl

a) Octadecanoic acid

1 2 3 4 5 6 7 O 6 5 4 3 2 1 O O OH n-hexadecanoic acid

OH b) Tridecanal

Fig. (110): Proposed pathway for the degradation of anthracene by B. amyloliquefaciens and it's mutant MAM-62 (4).

250 Results and Discussion

The proposed pathway for anthracene by B. amyloliquefaciens contains a set of metabolits not present in the other proposed pathways before, like thioxanthene and 7, 9 di tetra-butyl-1-oxospiro-4,5-deca-6,9- diene-2,8- dione and benzene hexachloro. Also for the mutant; like octadecanoic acid and tridecanal. These all previous intermediates are not found by (Ye et al., 2011) pathway by Aspergillus fumigatus in which the metabolites are Anthrone, Anthraquinone, and phthalic acid.

The results of pyrene degradation as determined by GC/MS analysis were indicated in Table (62) and Fig. (111). The results revealed that, non of the parent strain B. amyloliquefaciens MAM-62 intermediate was coinside with any intermediates of the mutant strain B. amyloliquefaciens MAM-62(4). Parent strain produced 4 intermediates, but mutant strain B. amyloliquefaciens MAM-62 (4) produced five intermediates and each pathway was different. Pyrene undergo oxidation and ring fission in a multi-steps to give benzeneethanol with more ring fission and polymerization of resulting aliphatic compounds give tetradecanoic acid. However, mutant strain MAM-62(4) undergo oxidation and ring fission to give methyl 2,3 di-o-acetyl-beta-D-xylopyranoside with more oxidation and ring fission produced (ethanol 2,2-butoxyethoxy).

251 Results and Discussion

Table (62): Intermediates determined by GC-MS analysis of pyrene degradation by B. amyloliquefaciens MAM-62 and its mutant MAM-62(4) after 24 hours incubation.

R.T MAM-62 Formula MAM-62(4) Formula

16.176 Benzene ethanol C8H10O - -

16.791 Hexanoic acid C9H18O2 - - 3,5,5’-trimethyl

17.336 2,4,6-cycloheptatri- C7H6O - - iene-1-one

18.252 - - Ethanol,2-(2-but- C8H18O3 oxyethoxy)-

18.853 - - Cyclopropane,2-(1,1- C12H22 dimethyl-2- pentenyl)1,1-diemthyl

22.403 - - Methyl2,3-di-o-acethyl- C10H16O7 B-D-xylopyranoside

31.713 - - Butanoic acid-3-methyl- C13H18O2 -2phenyl ethyl ester

32.468 - - Pentachlorophenol C6HCl5O

32.566 Tetradecanoic acid C14H28O2 - -

39.806 Pyrene C16H10 Pyrene C16H10

252 Results and Discussion

Pyrene MAM-62(4) MAM-62 1) Oxidation 2) Ring fission 1) Oxidation 2,4,6 cyclohetatrien 1-one 2) Ring fission 1) Oxidation O 2) Ring fission 3) Chloronation O O OH Butonic acid, 3-methyl 1) Oxidation 2-phenyl ethyl ester 2) Ring fission OH 3) Cyclopolymerizm Cl Cl Benzeneethanol O 1) Oxidation 2) Ring fission Cl Cl O 3) methylation 1) Ring fission Cl O OH 2) Polymerization Penta chloro O O phenol O O

OH Methyl 2,3 di-o-acetyl-beta-D-xylopyranoside Hexanoic acid 3,5,5-trimethyl

More oxidation O and ring fission

OH Tetradecanoic acid Cyclopropane 2(1,1 dimethyl 2-pentenyl 1,1 dimethyl)

O OH O Ethanol 2,2-butoxyethoxy Fig. (111): Proposed pathway for pyrene degradation by B. amyloliquefaciens MAM-62 and its mutant MAM-62 (4).

253 Results and Discussion

The proposed pathway for pyrene by B. amyloliquefaciens contains a set of metabolits not present in the other proposed pathways before. As indicated by (Cerniglia, 1992) for Mycobacterium sp., the intermediates are, phenanthroate, lead to cis cinnamate or phthalate or hydroxy perinaphtenone. Other pathway for (Dean-Ross and Cerniglia, 1996) by M. flavescens proposed a different set of metabolites from pyrene included cis pyrene dihydrodiol, phenanthrene dioic acid, phenanthroic acid, reaching to phthalate. The proposed pathway for bacterial consortium explained by (Luan et al., 2006) include cis pyrene dihydrodiol, cis dihydroxypyrene, lactone as well as hydroxy thenanthrene; and for fungus like Aspergillus niger, in the proposed pathway by Wunder et al. ( 1994); the metabolites were, pyrene oxide, hydroxy pryene oxide.

The results of GC/MS analys of the intermediate compounds resulted from benzo-a-anthracene degradation by the parent strain B. amyloliquefacies MAM-62 and its mutant B. amyloliquefacies MAM-62 (4) were indicated in Table (63) and Fig. (112), (113). The results revealed that parent strain MAM-62 produced 9 intermediates. However, the mutant strain MAM-62(4) produced 15 intermediates. Both the parent strain and its mutant produced a number of aliphatic acids (6 similar compounds) named Hexanoic acid, Hexanoic acid, 2- ethyl, hepatnoic acid, Octanoic acid, Nonanoic acid and n-hexadecanoic acid.

The corner stone in B-a-anthracene degradation was Benzo-a- anthracene 7, 12 dione which produced by the parent strain and its mutant.

254 Results and Discussion

Table (63): Intermediates determined by GC-MS analysis of benzo-a- anthracene degradation by B. amyloliquefaciens MAM-62 and its mutant MAM-62(4) after 24 hours incubation.

R.T MAM-62 Formula MAM-62(4) Formula

12.272 Hexanoic acid C6H12O2 Hexanoic acid C6H12O2

15.166 Hepatanoic acid C7H14O2 Heptanoic acid C7H14O2

16.184 Benzeneethanol C8H10O - -

16.289 Hexanoic acid,2- C8H16O2 Hexanoic acid, 2-ethyl C8H16O2 ethyl

17.553 - - N-1-(2-chloro-2- C10H20ClN ethylbutylidene)-T- butylamine

18.276 Octanoic acid C8H16O2 Octanoic acid C8H16O2

19.574 - - Propanamide, N-1(1,1 C9H19NO dimethyl)2,2-dimethyl

21.084 Nonanoic acid C9H18O2 Nonanoic acid C9H18O2

29.380 - - -Pheylethyl butyrate C12H16O2

31.348 - - 2,2-Dimethyl-N- C13H19NO phenethyl- propionamide

31.752 - - Butanoic acid, 3- C13H18O2 methyl,2-phenylethyl ester

33.841 Indol-5-aldhyde C9H7NO Indol-5-aldhyde C9H7No

36.666 n-Hexadecanoic C16H32O2 Hexadecanoic acid C16H32O2 acid

43.383 - - 4,4,8-trimthylnon-5- C12H22O enal

47.992 Benz(a)anthracene C18H10O2 Benz(a)anthracene7,12- C18H10O 7,12 dione dione

57.368 - - Sitosterol C29H50O

61.832 b-Sitosterol acetate C29H48 b-Sitosterol acetate C29H48

255 Results and Discussion

O

I- Hexanoic acid Benzo-a-anthracene oxidation

Oxidation O b-Sitosterol 1- Ring fission 1- Ring fission O O OH 2- Fussion 2- Polymerization 3-Side chain substitution II- Hexanoic acid, 2-ethyl O 1- Ring fission Benzo-a-anthracene 7,12 dione OH O 2- Indol ring O OH 2 3 4 5 7 formation O 1 6 III- Heptanoic acid 1- Ring fission NH HO VI- Hexadecanoic acid 2-Side chain substitution Indol-5-aldhyde OH OH O OH IV- Octanoic acid O Benzene ethanol V- Nonanoic acid Fig (112): Proposed pathway of benzo-a-anthracene degradation by B. amyloliquefaciens MAM-62.

256 Results and Discussion

O

OH I- Hexanoic acid

1- Ring fission 2- Fussion O OH Benzo-a-anthracene oxidation 3-Side chain substitution I- Hexanoic acid, 2-ethyl Oxidation O b-Sitosterol O acetate 1- Ring fission O OH III- Heptanoic acid 2- Polymerization 1- Ring fission 2- Indol ring formation O OH Benzo-a-anthracene 7,12 dione B-Sitosterol

VII OH O O IV- Octanoic acid HO Hexadecanoic acid 1- Ring fission 2-Side chain OH OH VI- 4,4,8 trimethyl- substitution O non-s-enal O V- Nonanoic acid NH Indol-5-aldhyde I II O III O O NH B-phenyl ethyl butrate O

III- 2,2-dimethyl-N-phenethyl Butanoic acid, propionamide 3-methyl ester Fig (113): Proposed pathway for benzo-a-anthracene degradation by the mutant of B. amyloliquefacians MAM-62(4).

257 Results and Discussion

The proposed pathway for B-a-Anth. by B. amyloliquefaciens contains a set of metabolites not present in other proposed pathways before.

A proposed pathway by (Cajthmal et al., 2006) includes a metabolites like B- a- quinone, phthalic acid, phthalic acide hydroxy, phthalic acid methylester, hydroxytetralone, naphthalone dione and dihydroxy naphthalene and the other indicated by (Schneider et al., 1996), like BAA dihydrodiol.

Generally, the previous results of this study clearly indicated that B. amyloliquefacians parent strain (MAM-62) was different from its mutant (MAM-62 (4) ) in their intermediates and pathways.

Also, the proposed pathways of B. amyloliquefacians are proved to be different and new compared with all proposed pathways mentioned by other investigators. .

258 Summary

SUMMARY

259 Summary

SUMMARY

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 (naphthalene-(Naph.); phenanthrene (Phen.); anthracene (Anth.); fluorancene (Flu.); acenaphthene (Acen.); pyrene (Pyr.) and benzo-a- anthracene (B-a-Anth.) as a sole carbon and energy source.

The growth of the seven different bacterial communities on the seven different PAHs had been investigated by recording their growth (O.D) and secretion of extracellular protein at zero time (initial) and after 7, 15, 21 and 28 days incubation.

The growth of community (1) on 50 mg/L Naph was 5.0, 10.0, 9.5 and 8.6 times the initial after 7, 15 ,21 and 28 days respectively. The same, community (1) gave the best growth on 250 mg/L Phen. (2.8, 2.7, 4.6 and 6.4 times the initial) after 7, 15, 21. and 28 days respectively. The ability of community (1) to grow on 50.0, 100 , 10.0 mg/L of Anth., Acen., and Flu., respectively, and 100 ug/L for both of Pyr. and B-a-Anth. have been recorded also.

The results revealed that community (1) gave the best growth and extracellular protein secretion followed by community (2). Count of these seven communities after 28 days incubation revealed an increase in bacterial count ranging from 0.8-1.0 ; 0.9-1.3 ; 0.9- 1.2 ;0.7 -1.0 ; 0.9-1.2 ; 0.9-1.3 and 0.6 – 1.2 log cycles on Naph., Phen. , Anth., Flu. , Acen., Pyr. And B-a-Anth. respectively.

The highest total bacterial count at zero time (initial) was 1.6 ҳ 107 CFU/ml for community (4), while the highest polycyclic aromatic hydrocarbon degrading bacterial (HDB) count was 3.0 ҳ 105 CFU/ml for community (3).

Six isolates (MAM-26, 29, 43, 62, 68, 78) were able to grow on different concentrations of five chosen PAHs (Naph., Phen., Anth., Pyr. And B-a-Anth). The bacterial isolates were previously isolated from soil polluted with petroleum oil, these isolates (MAM-26, 43, 62, 68 and 78). and standard strain Enterobacter cloacae MAM-4 were grown on five concentrations of naphthalene (Naph.) as a sole carbon and energy source. The abilities of these isolates to degrade Naph. have been investigated. The

260 Summary growth (O.D) and extracellular protein secretion were determined after 1,2,3,4,5,6,7,14 and 21 days incubation for each strain. Degradation of Naph. was quantified by High Performance Liquid Chromatography (HPLC). The results revealed that the best isolate was MAM-62 which degrades 78.0% , 83.0%,88% and 97.0% of 500 , 750, 1000 and 2000 mg/L of Naph. respectively.

The results of GC/MS analysis is indicated that B. amyloliquefaciens MAM-62 degraded naphthalene to give 23 intermediate compounds, meanwhile the mutant of B. amyloliquefaciens MAM-62(4) degraded naphthalene to give 9 intermediate compounds. Six intermediates compounds were found in both parent B. amyloliquefaciens MAM-62 and its mutant B. amyloliquefaciens MAM-62 (4). These compounds were Heptanoic acid, Hexanoic acid 2-ethyl, Nonanoic acid, 2-methyl indanone, Indol-5-aldhyde and Hexadecanonic acid.

Also in case of phenanthrene , The isolates (MAM-26 , 43 , 62 ,68 and 78), and standard strain Enterobacter cloacae MAM-4 were grown on five concentrations of phenanthrene (Phen.) as a sole carbon and energy source. The abilities of these isolates to degrade Phen. have been investigated. The growth (O.D) and extracellular protein secretion were determined after 1,2,3,4,5,7,14 and 21 days incubation for each strain. Degradation of Phen. was quantified by HPLC. The results revealed that isolate MAM-62 was the best one, degrades 89.0%, 70.0%,81% ,96% and 98.0% of 250 .500 , 750 , 1000 and 1500 mg/L of Phen. respectively.

Phenanthrene undergo oxidation to give 4(1H)-phenathrenone 2,3 dihydro with ring fission and more oxidation transformed to dimethylphathalate by B. amyloliquefaciens MAM-62. However, oxidation of phenanthrene by mutant B. amyloliquficans MAM-62(4) produced 1,2,3,4 teterhydro-phenanthrene -4-ol, phenanthrene-9-methoxy and 9-phenanthrenol. Further oxidation and ring fission produced hexanoic acid and hexanoic acid 2,ethyl (aliphatic acids).

The same for anthracene, these isolates (MAM-26 ,29, 43 , 62 ,68 and 78 ) and standard strain Enterobacter cloacae MAM-4 were grown on five concentrations of Anthracene (Anth.) as a sole carbon and energy source. The abilities of these isolates to degrade Anth. have been investigated . The growth (O.D) and extracellular protein secretion were determined after 1,2,3,4,5,7,14 and 21 days incubation for each strain. Degradation of Anth. was quantified by HPLC. The results revealed that isolate MAM-43 was the

261 Summary best one which degrades 80.5% , 96.0%, 62%, 78.5 and 79.0% of 40 , 50 , 75, 100 and 150 mg/L of Anth. respectively.

The results of GC/MS analysis of anthracene degradation by B. amyloliquefaciens revealed that, the parent strain B. amyloliquefaciens MAM-62 produced 6 intermediate compounds, while its mutant strain MAM-62(4) produced 4 intermediates. The cornstone in Anthracene degradation was napthalane which produced in case of both the parent strain and its mutant.

Also for pyrene, these isolates (MAM-26, 29, 62 and 68). and standard strain Enterobacter cloacae MAM-4 were grown on five concentrations of pyrene (Pyr.) as a sole carbon and energy source. The abilities of these isolates to degrade Pyr. have been investigated . The growth (O.D) and extracellular protein secretion were determined after 1,2,3,4,5,7,14 and 21 days incubation for each strain. Degradation of Pyr. was quantified by HPLC. The results revealed that isolate MAM-29 degrade 95.0%, 90.5% and 90.3% of 100, 200 and 300 ug/L of Pyr. respectively.

Pyrene degradation by B.amyloliquifaciens MAM-62 produced 4 intermediates after 24 hrs. incubation as determined by GC/ MS. These intermediates were benzeneethanol; hexanoic acid 3,5,5'-trimethyl;2,4,6- -1-one and tetradecanoic acid. Mutant of B.amyloliquifaciens MAM-62 (4) resulted from exposure to gamma radiation produced five different intermediates.

And for B-a-Anth., these isolates (MAM-26, 29, 62 and 68) and standard strain Enterobacter cloacae MAM-4 were grown on five concentrations of B-a-anthracene as a sole carbon and energy source. The abilities of these isolates to degrade B-a-Anth. have been investigated. The growth (O.D) and extracellular protein secretion were determined after 1,2,3,4,5,7,14 and 21 days incubation for each strain. The degradation of B- a-Anth. was quantified by HPLC. The results revealed thatbest isolate is MAM-26 which degrades 60.0%, 57.0%, 42.0%, 55.5 and 64.0% of 100, 200, 300, 400 and 500 ug/L of B-a-Anth., respectively.

The results of GC/MS analyses of the intermediate compounds resulted from benzo-a-anthracene degradation by the parent strain B. amyloliquefaciens MAM-62 and its mutant B. amyloliquefacies MAM-62 (4) indicated that the parent strain MAM-62 produced 9 intermediates. However, the mutant strain MAM-62(4) produced 15 intermediates. Both the parent strain and its mutant produced a number of aliphatic acids (6 similar

262 Summary compounds) named Hexanoic acid, Hexanoic acid, 2 ethyl, hepatnoic acid, Octanoic acid, Nonanoic acid and n-hexadecanoic acid.

The corner stone in B-a-anthracene degradation was Benzo-a- anthracene 7, 12 dione which produced by the parent strain and its mutant.

The best degraders bacterial isolates MAM-29 and MAM-62 were identified by 16S-rRNA. The isolated strain MAM-29 showed 100% similarity with Achromobacter xylosoxidans strain R8-558 with accession No. JQ 659958.1 So isolate MAM-29 was identified as Achromobacter xylosoxidans with accession No. JN 038055. However MAM-62 showed 99% similarity with Bacillus sonorensis AJ 586363 with accession No. DQ 350821. So isolate MAM-62 was identified as Bacillus amyloliqueficiens with accession No. JN 038054.

The most promising bacterial strain MAM-62 have been exposed to different doses of gamma radiation. As the dose of γ- radiation increased, the viable count of MAM-62 decreased. Dose of 15 KGy reduced the viable count by 7.25 log cycles.

263 References

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304

ARABIC SUMMARY

اﻟﻤﻠـﺨـﺺ اﻟﻌـﺮﺑــﻰ

ﻭﺠﺩ ﺃﻥ ﺴﺒﻌﺔ ﻤﻥ ﺍﻟﻤﺠﺘﻤﻌﺎﺕ ﺍﻟﺒﻜﺘﻴﺭﻴﺔ ﺍﻟﻤﺨﺘﻠﻔﺔ ﺍﻟﻤﻌﺯﻭﻟﺔ ﻤـﻥ ﺍﻻﺭﺍﻀـﻰ ﺍﻟﻤﻠﻭﺜـﺔ ﺒﺎﻟﺒﺘﺭﻭل ﻤﻥ ﺸﺭﻜﺔ ﺍﻟﻘﺎﻫﺭﺓ ﺍﻟﻜﺒﺭﻯ ﻟﺘﻜﺭﻴﺭ ﺍﻟﺒﺘﺭﻭل ﺒﺎﻟﻘﻠﻴﻭﺒﻴﺔ , ﻜﺎﻨﺕ ﻟﻬﺎ ﺍﻟﻘﺩﺭﺍﺕ ﺍﻟﻤﺨﺘﻠﻔﺔ ﻋﻠﻰ ﺍﻟﻨﻤﻭ ﻋﻠﻰ ﻤﺭﻜﺒﺎﺕ ﺍﺭﻭﻤﺎﺘﻴﺔ ﻋﺩﻴﺩﺓ ﺍﻟﺤﻠﻘﺎﺕ . ﻭﻗﺩ ﺘﻡ ﺘﻘﺩﻴﺭ ﻗﻴﺎﺱ ﺍﻟﻨﻤﻭ ﻭ ﺍﻓﺭﺍﺯ ﺍﻟﺒﺭﻭﺘﻴﻨﺎﺕ ﻓﻰ ﺍﻟﻭﺴﻁ ﻋﻨﺩ ﺍﻟﺒﺩﺍﻴﺔ ﻭ ﺒﻌﺩ ﻓﺘﺭﺍﺕ ﺘﺤﻀﻴﻨﻴﺔ ﻤﺨﺘﻠﻔﺔ (7’15’21 ﻭ 28 ﻴﻭﻡ).

ﻭﻗﺩ ﺍﺜﺒﺘﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺍﻥ ﻨﻤﻭ ﺍﻟﻤﺠﺘﻤﻊ ﺍﻟﺒﻜﺘﻴﺭﻯ(1) ﻋﻠﻰ 500 ﻤﻠﻠىﺠﺭﺍﻡ/ﻟﺘﺭ ﻤﻥ ﺍﻟﻨﻔﺘﺎﻟﻴﻥ ﻜﺎﻥ 5.0، 9.5،10.0 ﻭ 8.6 ﻤﺭﺍﺕ ﻋﻥ ﺍﻟﺒﺩﺍﻴﺔ ﻟﺔ ﺒﻌﺩ 21،15،7 ﻭ28 ﻴﻭﻡ ﺒﺎﻟﺘﺘﺎﺒﻊ.

ﻭ ﺍﻴﻀﺎ ﺍﻟﻤﺠﺘﻤﻊ ﺍﻟﺒﻜﺘﻴﺭﻯ(1) ﺍﻋﻁﻰ ﺍﻓﻀل ﻨﻤﻭ ﻋﻠﻰ 250 ﻤﻠﻠىﺠﺭﺍﻡ/ﻟﺘـﺭ ﻤـﻥ ﺍﻟﻔﻴﻨـﺎﻨﺜﺭﻴﻥ (4.6،2.7،2.8 ﻭ 6.4 ﻤﺭﺍﺕ ﻋﻥ ﺍﻟﺒﺩﺍﻴﺔ) ﺒﻌﺩ 21،15،7 ﻭ28 ﻴﻭﻡ ﺒﺎﻟﺘﺘﺎﺒﻊ.

ﻭﺍﻴﻀﺎ ﻋﻴﻥ ﻤﻘﺩﺭﺓ ﺍﻟﻤﺠﺘﻤﻊ ﺍﻟﺒﻜﺘﻴﺭﻯ(1) ﻋﻠﻰ 100.0،50.0ﻭ10.0 ﻤﻠﻠىﺠﺭﺍﻡ/ﻟﺘـﺭ ﻟﻼﻨﺜﺭﺍﺴﻴﻥ ﻭ ﺍﻻﺴﻴﻨﻔﺜﻴﻥ ﻭ ﺍﻟﻔﻠﻭﺭﺍﻨﺜﻴﻥ ﺒﺎﻟﺘﺘﺎﺒﻊ ﻭ 100ﻤﻴﻜﺭﻭﺠﺭﺍﻡ /ﻟﺘﺭ ﻟﻜـ ﻼ ﻤـﻥ ﺍﻟﺒﻴـﺭﻴﻥ ﻭ ﺍﻟﺒﻨﺯﻭ-ﺍ-ﺍﻨﺜﺭﺍﺴﻴﻥ .

ﻭﻤﻥ ﺍﻟﻨﺘﺎﺌﺞ ﻭﺠﺩ ﺍﻥ ﺍﻟﻤﺠﺘﻤﻊ ﺍﻟﺒﻜﺘﻴﺭﻯ(1) ﺍﻋﻁﻰ ﺍﻓﻀل ﻨﻤﻭ ﻭ ﺍﻨﺘﺎﺝ ﺒﺭﻭﺘﻴﻨﻰ ﻴﻠﻴـﺔ ﺍﻟﻤﺠﺘﻤﻊ ﺍﻟﺒﻜﺘﻴﺭﻯ(2).

ﺘﻡ ﺘﻌﻴﻴﻥ ﺍﻟﻌﺩﺩ ﻟﻬﺫﺓ ﺍﻟﻤﺠﻤﻭﻋﺎﺕ ﺍﻟﻤﻴﻜﺭﻭﺒﻴﺔ ﺒﻌﺩ 28 ﻴﻭﻡ ﺘﺤﻀﻴﻥ.ﻭﻭﺠﺩ ﺍﻥ ﺍﻟﺯﻴﺎﺩﺓ ﻓﻰ ﺍﻟﻌﺩﺩ ﺍﻟﺒﻜﺘﻴﺭﻯ ﻟﻬﺫﺓ ﺍﻟﻤﺠﻤﻭﻋﺎﺕ ﺍﻟﺴﺒﻌﺔ ﻴﺘـﺭﺍﻭﺡ ﻤـﻥ - -0.8 1.0, 1.3-0.9 , 1.2-0.9 ,0.7-1.0 ,0.9-1.2 ﻋﻠــﻰ ,0.9-1.3 ﻭ 0.6-1.2 ﺩﻭﺭﺓ ﻟﻭﻏﺎﺭﻴﺘﻤﻴــﺔﻋﻠﻰ . ﺍﻟﻨﻔﺘــﺎﻟﻴﻥ, ﺍﻟﻔﻴﻨﻨﺜﺭﻴﻥ, ﺍﻻﻨﺜﺭﺍﺴﻴﻥ, ﺍﻟﻔﻠﻭﺭﺍﻨﺴﻴﻥ, ﺍﻻﺴﻴﻨﻔﺜﻴﻥ, ﺍﻟﺒﻴﺭﻴﻥ ﻭ ﺍﻟﺒﻨﺯﻭ- ﺍ- ﺍﻨﺜﺭﺍﺴﻴﻥ ﺒﺎﻟﺘﺘﺎﺒﻊ.

7 ﻭﻭﺠﺩ ﺍﻥ ﺍﻋﻠﻰ ﻋﺩﺩ ﺒﻜﺘﻴﺭﻯ ﺍﻟﻜﻠﻰ ﻋﻨﺩ ﺍﻟﺒﺩﺍﻴﺔ ﻟﻔﺘﺭﺓ ﺍﻟﺘﺤﻀﻴﻥ ﻜﺎﻥ 1.6 * 10 ﻭﺤﺩﺓ ﻤﺴﺘﻌﻤﺭﺓ ﺒﻜﺘﻴﺭﻴﺔ /ﻤﻠﻠﻰ ﻟﻠﻤﺠﻤﻭﻋﺔ ﺍﻟﺒﻜﺘﻴﺭﻴﺔ ﺭﻗﻡ (4) ﻭﺍﻟﻌﺩﺩ ﺍﻟﺒﻜﺘﻴـﺭﻯ ﺍﻟﻤﻜﺴـﺭ ﻟﻠﻤﺭﻜﺒـﺎﺕ 5 ﺍﻻﺭﻭﻤﺎﺘﻴﺔ ﻋﺩﻴﺩﺓ ﺍﻟﺤﻠﻘﺎﺕ 3.0* 10 ﻭﺤﺩﺓ ﻤﺴﺘﻌﻤﺭﺓ ﺒﻜﺘﻴﺭﻴﺔ /ﻤﻠﻠﻰ. ﻟﻠﻤﺠﻤﻭﻋﺔ ﺍﻟﺒﻜﺘﻴﺭﻴﺔ ﺭﻗـﻡ .(3)

ﻭﺠﺩ ﺍﻥ ﺴﺕ ﻋﺯﻻﺕ (MAM-26, 29, 43, 62, 68, 78) ﻜﺎﻥ ﻟﻬﺎ ﺍﻟﻤﻘﺩﺭﺓ ﻋﻠـﻰ ﺍﻟﻨﻤﻭ ﻋﻠﻰ ﺘﺭﻜﻴﺯﺍﺕ ﻤﺨﺘﻠﻔﺔ ﻤﻥ ﺨﻤﺱ ﻤﻥ ﺍﻟﻤﺭﻜﺒﺎﺕ ﺍﻻﺭﻭﻤﺎﺘﻴﺔ ﻋﺩﻴـﺩﺓ ﺍﻟﺤﻠﻘـﺎﺕ (ﻨﻔﺘـﺎﻟﻴﻥ , ﻓﻴﻨﻨﺜﺭﻴﻥ, ﺍﻨﺜﺭﺍﺴﻴﻥ, ﺒﻴﺭﻴﻥ ﻭ ﺒﻨﺯﻭ- ﺍ- ﺍﻨﺜﺭﺍﺴﻴﻥ).

ﻫﺫﺓ ﺍﻟﺴﻼﻻﺕ ﺍﻟﺴﺎﺒﻕ ﺫﻜﺭﻫﺎ ﻤﻊ ﺍﻟﻌﻴﻨﺔ ﺍﻟﻘﻴﺎﺴﻴﺔ ﺍﻨﺘﻴﺭﻭﺒﺎﻜﺘﺭ ﻜﻠﻭﺍﻜﺎ MAM-4 ﻨﻤـﺕ ﻋﻠﻰ ﺨﻤﺱ ﺘﺭﻜﻴﺯﺍﺕ ﻤﺨﺘﻠﻔﺔ ﻤﻥ ﻜﻤﺼﺩﺭ ﻭﺤﻴﺩ ﻟﻠﻜﺭﺒﻭﻥ ﻭ ﺍﻟﻁﺎﻗﺔ.

ﺘﻡ ﺘﻌﻴﻴﻥ ﻤﻘﺩﺭﺓ ﻫﺫﺓ ﺍﻟﺴﻼﻻﺕ ﻟﺘﻜﺴﻴﺭ ﺍﻟﻨﻔﺜﺎﻟﻴﻥ ﻭ ﺫﻟﻙ ﻋﻥ ﻁﺭﻴﻕ ﺘﻘﺩﻴﺭ ﺍﻟﻨﻤﻭ (ﺍﻟﻜﺜﺎﻓﺔ ﺍﻟﻀﻭﺌﻴﺔ) ﻭﺍﻟﺒﺭﻭﺘﻴﻥ ﺍﻟﻤﻨﺘﺞ ﺨﺎﺭﺠﻴـﺎ ﻟﻠﺨﻼﻴـﺎ ﺍﻟﻤﻴﻜﺭﻭﺒﻴـﺔ ﻭ ﻋـﺩﺩ ﺍﻟﺨﻼﻴـﺎ ﻋﻨـﺩ ﻓﺘـﺭﺍﺕ ﺍﻟﺘﺤﻀﻴﻥ1,2,3,4,5,6,7,14.21 ﻴﻭﻡ ﻟﻜل ﺴﻼﻟﺔ ﻜﻤﺎ ﺘﻡ ﺘﻘﺩﻴﺭ ﺘﻜﺴﻴﺭ ﺍﻟﻨﻔﺜﺎﻟﻴﻥ ﻋـﻥ ﻁﺭﻴـﻕ HPLC ﻭﻤﻥ ﺍﻟﻨﺘﺎﺌﺞ ﻨﺠﺩ ﺍﻥ ﺘﻜﺴﻴﺭ ﺍﻟﺴﻼﻟﺔ MAM- 26 ﻟﻠﻨﻔﺜﺎﻟﻴﻥ ﻜﺴﺭﺕ 88 ,83.0 ,78.0 and 0,97.0% ﻟﻠﺘﺭﻜﻴﺯﺍﺕ 500ﻭ750ﻭ1000ﻭ2000 ﻤﻠﻠﻰ ﺠﺭﺍﻡ/ﻟﺘﺭ ﻤﻥ ﺍﻟﻨﻔﺜﺎﻟﻴﻥ ﺒﺎﻟﺘﺘـﺎﺒﻊ .ﺍﻴﻀﺎ ﻓﻰ ﺤﺎﻟﺔ ﺍﻟﻔﻴﻨﻨﺜﺭﻴﻥ ﻟﻠﺴﻼﻻﺕ MAM-26,43,62,68 and 78 ﻫﺫﺍ ﻤﻊ ﺍﻟﺴﻼﻟﺔ ﺍﻟﻘﻴﺎﺴﻴﺔ ﺍﻨﺘﻴﺭﻭﺒﺎﻜﺘﺭ ﻜﻠﻭﺍﻜﺎ MAM-4 ﻨﻤﺕ ﻋﻠﻰ ﺘﺭﻜﻴﺯﺍﺕ ﻤﺨﺘﻠﻔﺔ ﻤﻨﺔ ﻜﻤﺼﺩﺭ ﻜﺭﺒﻭﻥ ﻭ ﻁﺎﻗﺔ ﻭﺤﻴﺩ ﻭ ﺘﻡ ﺘﻘﺩﻴﺭ ﺍﻟﻨﻤﻭ ﻭ ﺍﻨﺘﺎﺝ ﺍﻟﺒﺭﻭﺘﻴﻥ ﺍﻟﺨﺎﺭﺠﻰ ﺒﻌﺩ 1ﻭ2ﻭ3ﻭ4ﻭ5ﻭ6ﻭ7ﻭ14ﻭ21 ﻴﻭﻡ ﺘﺤﻀﻴﻥ ﻭ ﺘـﻡ ﺘﻘﺩﻴﺭ ﺘﻜﺴﻴﺭ ﺍﻟﻔﻴﻨﻨﺜﺭﻴﻨﺒﻭﺍﺴﻁﺔ ﺍﻟﺨﻤﺱ ﺴﻼﻻﺕ ﻭﻭﺠﺩ ﺍﻥ ﺍﻟﺴﻼﻻﺓ MAM-62 ﻜﺎﻨﺕ ﺍﻓﻀﻠﻬﻤﻭ ﻨﺘﺎﺌﺠﻬﺎ ﻫﻰ % (and 98.0 96.0 ,81.0 ,70.0 ,89.0)

ﻋﻠﻰ ﺍﻟﺘﺭﻜﻴﺯﺍﺕ 250ﻭ500ﻭ750 1000 ﻭ1500 ﻤﻠﻠىﺠﺭﺍﻡ /ﻟﺘﺭ ﺒﺎﻟﺘﺘﺎﺒﻊ ﻭ ﺍﻴﻀـﺎ ﻭﺠﺩ ﺍﻥ ﻨﺘﺎﺌﺞ ﺘﻜﻴﺴﺭ ﺍﻟﻁﻔﺭﺓ ﻤﺨﺘﻠﻑ ﻋﻥ ﺍﻟﺴﻼﻻﺓ ﺍﻻﻡ.

ﺍﻴﻀﺎ ﻟﻤﺭﻜﺏ ﺍﻻﻨﺜﺭﺍﺜﻴﻥ ﺍﻟﺴﻼﻻﺕ MAM-26, 29, 43,62,68 and 78 ﻤﻊ ﺍﻟﺴﻼﻟﺔ ﺍﻟﻘﻴﺎﺴﻴﺔ ﺍﻨﺘﻴﺭﻭﺒﺎﻜﺘﺭ ﻜﻠﻭﺍﻜﺎ MAM-4 ﻜﻤﺎ ﺴﺒﻕ ﻭﻤﻥ ﺍﻟﻨﺘﺎﺌﺞ ﻭﺠﺩ ﺍﻥ ﺍﻟﺴﻼﻟﺔ MAM-43 ﻫﻰ ﺍﻻﻓﻀل ﺤﻴﺙ ﻜﺴﺭﺕ 80.5-96.0-62.0-78.5-79.5 % ﻟﻠﺘﺭﻜﻴﺯﺍﺕ 150-100-75-50-40 ﻤﻠﻠىﺠﺭﺍﻡ/ﻟﺘﺭ ﺒﺎﻟﺘﺘﺎﺒﻊ.

ﺍﻴﻀﺎ ﻟﻠﺒﻴﺭﻴﻥ ﺍﻟﺴﻼﻻﺕ MAM-26, 29,62,68 ﻤﻊ ﺍﻟﺴﻼﻟﺔ ﺍﻟﻘﻴﺎﺴﻴﺔ ﺍﻨﺘﻴﺭﻭﺒـﺎﻜﺘﺭ ﻜﻠﻭﺍﻜﺎ MAM-4 ﻨﻤﺕ ﻋﻠﻰ ﺘﺭﻜﻴﺯﺍﺕ ﻤﺨﺘﻠﻔﺔ ﻤﻥ ﺍﻟﻤﺭﻜﺏ ﻜﻤﺼﺩﺭ ﻜﺭﺒﻭﻥ ﻭ ﻁﺎﻗﺔ ﻭﺤﻴﺩ

ﺘﻡ ﺘﻌﻴﻴﻥ ﻗﺩﺭﺓ ﻫﺫﺓ ﺍﻟﻌﺯﻻﺕ ﻟﺘﻜﺴﻴﺭ ﺍﻟﺒﻴﺭﻴﻥ ﻭ ﺫﻟﻙ ﻋﻥ ﻁﺭﻴﻕ ﺘﻘﺩﻴﺭ ﺍﻟﻨﻤـﻭ (ﺍﻟﻜﺜﺎﻓـﺔ ﺍﻟﻀﻭﺌﻴﺔ) ﻭ ﺍﻟﺒﺭﻭﺘﻴﻥ ﺍﻟﻤﻨﺘﺞ ﺨﺎﺭﺠﻴﺎ ﻟﻠﺨﻼﻴﺎ ﺍﻟﻤﻴﻜﺭﻭﺒﻴﺔ ﻭﻋﺩﺩ ﺍﻟﺨﻼﻴﺎ ﻋﻨﺩ ﻓﺘﺭﺍﺕ ﺍﻟﺘﺤﻀﻴﻥ -1 2-3-4-5-6-7-14 ﻭ 21 ﻴﻭﻡ ﻟﻜل ﺴﻼﻟﺔ .

ﻜﻤﺎ ﺘﻡ ﺘﻘﺩﻴﺭ ﺘﻜﺴﻴﺭ ﺍﻟﺒﻴﺭﻴﻥ ﻋﻥ ﻁﺭﻴﻕ (HPLC) ﻭﻤﻥ ﺍﻟﻨﺘﺎﺌﺞ ﻨﺠﺩ ﺍﻥ ﺘﻜﺴﻴﺭ ﺍﻟﺴﻼﻟﺔ MAM-29 ﻟﻠﺒﻴﺭﻴﻥ ﻋﻨﺩ ﺍﻟﺘﺭﻜﻴﺯﺍﺕ ug/l 100,200,300 ﻜﺎﻨﺕ 95.0%,90.5%ﻭ%90.3 ﺒﺎﻟﺘﺘﺎﺒﻊ ﻭﻫﺫﺍ ﺍﻓﻀل ﺘﻜﺴﻴﺭ ﻟﻬﺫﺓ ﺍﻟﺴﻼﻟﺔ ﺒﺎﻟﻤﻘﺎﺭﻨﺔ ﺒﺎﻟﺴﻼﻻﺕ ﺍﻻﺨﺭﻯ .ﻭﻫﺫﺓ ﺍﻟﺴﻼﻟﺔ ﻋﺭﻓﺕ ﺒﺎل .16S-rRNA

ﺍﻴﻀﺎ ﻤﺜل ﻤﺎ ﺴﺒﻕ ﻟﻠﺒﻨﺯﻭ ﺍ ﺍﻨﺜﺭﺍﺴﻴﻥ ﺍﻟﺴﻼﻻﺕ MAM-26, 29,62,68 ﻤﻊ ﺍﻟﺴﻼﻟﺔ ﺍﻟﻘﻴﺎﺴﻴﺔ ﺍﻨﺘﻴﺭﻭﺒﺎﻜﺘﺭ ﻜﻠﻭﺍﻜﺎ MAM-4 ﻨﻤﺕ ﻋﻠﻰ ﺘﺭﻜﻴﺯﺍﺕ ﻤﺨﺘﻠﻔﺔ ﻤﻥ ﺍﻟﻤﺭﻜﺏ ﻜﻤﺼﺩﺭ ﻜﺭﺒﻭﻥ ﻭ ﻁﺎﻗﺔ ﻭﺤﻴﺩ ﻭﻭﺠﺩ ﻤﻥ ﻨﺘﺎﺌﺞ ﺍل(HPLC) ﺍﻥ MAM-26 ﻫﻰ ﺍﻻﻓﻀل ﻭ ﻜﺴﺭﺕ ﺍﻟﻤﺭﻜـﺏ ﺒﻨﺴﺒﺔ 60-57-42-55.5 – 64 % ﻟﻠﺘﺭﻜﻴﺯﺍﺕ 100-200-300-400-500 ﻤﻴﻜﺭﻭﺠﺭﺍﻡ ﻟﻜل ﻟﺘﺭ ﺒﺎﻟﺘﺘﺎﺒﻊ. .ﻤﻤﺎ ﺴﺒﻕ ﻭﺠﺩ ﺍﻥ ﺍﻓﻀل ﺴﻼﻟﺘﻴﻥ ﻫﻤﺎ MAM-26 ﻭ MAM-62. ﻭ ﻗﺩ ﺘﻡ ﺘﻌﺭﻴﻑ ﻟﻬﺎﺘﺎﻥ ﺍﻟﺴﻼﻟﺘﺒﻥ ﺒﺎﻟﺘﻘﻨﻴﺔ ﺍﻟﺠﻴﻨﻴﺔ ( 16S-rRNA) ﻭ ﻗﺩ ﺘﻡ ﺘﺤﺩﻴﺩ ﺍﻟﺘﺘﺎﺒﻊ ﺍﻟﺠﻴﻨـﻰ ﻭ ﻋﻤل ﺸﺠﺭﺓ ﺍﻟﺘﺸﺎﺒﺔ ﻟﻬﻤﺎ ﻭ ﻗﺩ ﻴﻭﺠﺩ ﺘﺸﺎﺒﺔ ﺒﻨﺴﺒﺔ 100% ﻟل MAM-26 ﻤـﻊ ﺴـﻼﻟﺔ ﺍل ﺍﻜﺭﻭﻤﻭﺒﺎﻜﺘﺭﺯﻴﻠﻭﺯﻭﻜﺴﻴﺩﺍﻨﺱ ﺴﻼﻟﺔ R8-558 ﺒﺭﻗﻡ ﺘﺘﺎﺒﻊ (JQ 659958.1) ﻟـﺫﻟﻙ ﺍﻟﺴـﻼﻟﺔ MAM-26 ﺘﹸﻌﺭﻑ ﺒﺎﻨﻬﺎ ﺍﻜﺭﻭﻤﻭﺒﺎﻜﺘﺭ ﺯﻴﻠﻭﺯﻭﻜﺴﻴﺩﺍﻨﺱ ﺒﺭﻗﻡ(JN038055). ﻭ ﺍﻴﻀﺎ ﻟﻠﺴﻼﻟﺔ MAM-62 ﺒﺎﻨﻬﺎ ﺒﺎﺴﻴﻠﺱ ﺍﻤﻴﻠﻭﻟﻜﻭﺍﻓﻴﻜﺎﻨﺱ ﺘﺤﺕ ﺭﻗﻡ ﺘﺘﺎﺒﻌﻰ JN038054 ﻭ ﻋﻤﻠـﺕ ﺸـﺠﺭﺓ ﺍﻟﺘﺸﺎﺒﺔ ﻟﻬﻤﺎ .

ﻋﺭﻀﺕ ﺍﻟﺴﻼﻟﺔ MAM-62 ﻟﺠﺭﻋﺎﺕ ﺍﺸﻌﺎﻋﻴﺔ ﻭﻭﺠﺩ ﻤﻥ ﺍﻟﻤﻨﺤﻨﻰ ﺍﻻﺸﻌﺎﻋﻰ ﺍﻨـﺔ ﻜﻠﻤﺎ ﺯﺍﺩﺕ ﺍﻟﺠﺭﻋﺔ ﺍﻻﺸﻌﺎﻋﻴﺔ ﻗل ﺍﻟﻌﺩﺩ ﺍﻟﺤﻰ ﻤﻥ ﺍﻟﺒﻜﺘﻴﺭﻴﺎ . ﻭﻭﺠﺩ ﺍﻥ ﺍﻟﺠﺭﻋﺔ KGy 15 ﺘﻘﻠل ﺍﻟﻌﺩﺩ ﺍﻟﺒﻜﺘﻴﺭﻯ ﺍﻟﻰ 7.25 ﺩﻭﺭﺓ ﻟﻭﻏﺎﺭﻴﺘﻤﻴﺔ .

ﻜﻤﺎ ﺘﻡ ﺩﺭﺍﺴﺔ ﺨﻁﻭﺍﺕ ﺘﻜﺴﻴﺭ ﺍﻟﻨﻔﺜﺎﻟﻴﻥ ﺒﻭﺍﺴﻁﺔ ﺴﻼﻟﺔ ﺍﻟﺒﺎﺴﻴﻠﺱ ﺍﻤﻴﻠـﻭ ﻟﻜﻭﺍﻓﻴﻜـﺎﻨﺱ MAM-26 ﻭ ﺍﻟﺘﻲ ﺘﻡ ﺘﻌﺭﻴﻔﻬﺎ ﻭ ﺒﻴﻥ ﺍﻓﻀل ﻁﻔﺭﺓ ﻨﺎﺘﺠﺔ ﻤﻨﻬﺎ ﻭﻭﺠﺩ ﻤﻥ ﻨﺘﺎﺌﺞ ﺍلGC/MS ﺍﻥ ﺍﻟﺴﻼﻟﺔ ﺍﻻﻡ ﺍﻨﺘﺠﺕ 23 ﻤﺭﻜﺏ ﻭﺴﻁ ﺒﻴﻨﻤﺎ ﺍﻟﻁﻔﺭﺓ ﻨﺘﺞ ﻋﻨﻬﺎ 9 ﻤﺭﻜﺒﺎﺕ ﻭ ﺴﻁﻴﺔ ﻓﻘﻁ ﻭ ﺴﺕ ﻤﻥ ﻫﺫﺓ ﺍﻟﻤﺭﻜﺒﺎﺕ ﻭﺠﺩ ﺍﻨﻬﺎ ﻤﺸﺘﺭﻜﺔ ﺒﻴﻥ ﺍﻟﺴﻼﻟﺔ ﺍﻻﻡ ﻭ ﺍﻟﻁﻔﺭﺓ ﺍﻟﻨﺎﺘﺠﺔ ﻤﻨﻬﺎ.

ﻭﺍﻴﻀﺎ ﻤﻥ ﻨﺘﺎﺌﺞ ﻟﻠﻔﻴﻨﺎﻨﺜﺭﻴﻥ GC/MS ﻭﺠﺩ ﺍﻥ ﺍﻟﻤﻭﻜﺒﺎﺕ ﺍﻟﻭﺴـﻁﻴﺔ ﻟﻠﺴـﻼﻟﺔ ﺍﻻﻡ ﻭ ﻤﺨﺘﻠﻔﺔ ﻋﻥ ﺍﻟﻨﺎﺘﺠﺔﻤﻨﻬﺎ ﺒﺎﻟﺘﻁﻔﺭ.ﻭ ﻫﺫﺍ ﺍﻴﻀﺎ ﻟﻼﻨﺜﺭﺍﺴﻴﻥ. ﻜﻤﺎ ﺘﻤﺕ ﺍﻟﻤﻘﺎﺭﻨﺔ ﺒﻴﻥ ﺨﻁﻭﺍﺕ ﻭ ﻨﻭﺍﺘﺞ ﺘﻜﺴﻴﺭ ﺍﻟﺒﻴﺭﻴﻥ ﻟﻠﺴﻼﻟﺔ ﺍﻻﺼﻠﻴﺔ ﻭ ﺍﻟﻁﻔﺭﺓ ﺍﻟﻨﺎﺘﺠﺔ ﻤﻥ ﺍﻟﺘﻌﺭﺽ ﻻﺸـﻌﺔ ﺠﺎﻤـﺎ . ﻋـﻥ ﻁﺭﻴـﻕ (GC/MS) ﻭ ﻗﺩ ﺍﺜﺒﺘﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺍﻥ ﺨﻁﻭﺍﺕ ﺍﻟﺘﻜﺴﻴﺭ ﻜﺎﻨﺕ ﻤﺨﺘﻠﻔﺔ ﻜﻠﻴﺎ ﻤﺎ ﺒﻴﻥ ﺍﻟﺴﻼﻟﺔ ﺍﻻﺼﻠﻴﺔ ﻭ ﺍﻟﻁﻔﺭﺓ. ﻭﻨﺘﻴﺠﺔ ﺍل(GC/MS) ﻟﻠﺒﻨﺯﻭﺍ ﺍﻨﺜﺭﺍﺴﻴﻥ ﺍﻴﻀﺎ ﺍﺜﺒﺘﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺍﻥ ﺨﻁﻭﺍﺕ ﺍﻟﺘﻜﺴـﻴﺭ ﻜﺎﻨﺕ ﻤﺨﺘﻠﻔﺔ ﻜﻠﻴﺎ ﻤﺎ ﺒﻴﻥ ﺍﻟﺴﻼﻟﺔ ﺍﻻﺼﻠﻴﺔ ﻭ ﺍﻟﻁﻔﺭﺓ.

اﻟﻤﺴﺘﺨﻠﺺ

ﺃﺳﻢ ﺍﻟﻄﺎﻟﺐ: ﻋﺒﲑ ﻣﻌﻮﺽ ﺑﺮﺗﻼ ﺟﺮﺟﺲ ﻋﻨﻮﺍﻥ ﺍﻟﺮﺳﺎﻟﺔ: ﺍﻟﺘﻜﺴﲑ ﺍﳊﻴﻮﻯ ﻟﻠﻤﺮﻛﺒﺎﺕ ﺍﻻﺭﻭﻣﺎﺗﻴﺔ ﻣﺘﻌﺪﺩﺓ ﺍﳊﻠﻘﺎﺕ ﺍﳍﻴﺪﺭﻭﻛﺮﺑﻮﻧﻴﺔ ﰱ ﺯﻳـﺖ ﺍﻟﺒﺘﺮﻭﻝ ﺍﳌﻠﻮﺛﺔ ﻟﻠﺒﻴﺌﺔ ﺍﻟﺪﺭﺟﺔ: ﺍﻟﺪﻛﺘﻮﺭﺍﺓ (ﺍﳌﻴﻜﺮﻭﺑﻴﻮﻟﻮﺟﻰ)

ﻋﯿﻨﺎت ﻣﻦ اﻟﺘﺮﺑﺔ ﻋﺰﻟﺖ ﻣﻦ اراﺿﻰ ﻣﻠﻮﺛﺔ ﺑﺎﻟﺒﺘﺮول ﻣﻦ ﺷﺮﻛﺔ اﻟﻘﺎھﺮة اﻟﻜﺒﺮى ﻟﺘﻜﺮﯾﺮ اﻟﺒﺘﺮول ﺑﺎﻟﻘﻠﯿﻮﺑﯿﺔ ھﺬة اﻟﻌﯿﻨﺎت ﻧﻤﻮا ﻋﻠﻰ ﺳﺒﻊ ﻣﺮﻛﺒﺎت اروﻣﺎﺗﯿﺔ ﻋﺪﯾﺪة اﻟﺤﻠﻘﺎت ، وﺟﺪ ان ﺳﺖ ﻋﺰﻻت (MAM-26, 29, 43, 62, 68, 78) ﻛﺎن ﻟﮭﺎ اﻟﻤﻘﺪرة ﻋﻠﻰ اﻟﻨﻤﻮ ﻋﻠﻲ ﺗﺮﻛﯿﺰات ﻣﺨﺘﻠﻔﺔ ﻣﻦ ﺧﻤﺲ ﻣﻦ اﻟﻤﺮﻛﺒﺎت اﻻروﻣﺎﺗﯿﺔ ﻋﺪﯾﺪة اﻟﺤﻠﻘﺎﺗﻮﺟﺪ ان اﻓﻀﻞ ﺳﻼﻟﺘﯿﻦ ھﻤﺎ MAM-26 و .MAM-62

و ﻗﺪ ﺗﻢ ﺗﻌﺮﯾﻒ ﻟﮭﺎﺗﺎن اﻟﺴﻼﻟﺘﺒﻦ ﺑﺎﻟﺘﻘﻨﯿﺔ اﻟﺠﯿﻨﯿﺔ (16S-rRNA) و ﻗﺪ ﺗﻢ ﺗﺤﺪﯾﺪ اﻟﺘﺘﺎﺑﻊ اﻟﺠﯿﻨﻰ و ﻋﻤﻞ ﺷﺠﺮة اﻟﺘﺸﺎﺑﺔ ﻟﮭﻤﺎ و ﻗﺪ ﻋﺮﻓﻮا اﻛﺮوﻣﻮﺑﺎﻛﺘﺮ زﯾﻠﻮزوﻛﺴﯿﺪاﻧﺲ وﺑﺎﺳﯿﻠﺲ اﻣﯿﻠﻮﻟﻜﻮاﻓﯿﻜﺎﻧﺲ ﺑﺎﻟﺘﺘﺎﺑﻊ. ﻋﺮﺿﺖ اﻟﺴﻼﻟﺔ MAM-62 ﻟﺠﺮﻋﺎت اﺷﻌﺎﻋﯿﺔ ﻟﺘﺤﺴﯿﻦ ﻗﺪرة اﻟﺘﻜﺴﯿﺮ ﻟﺪﯾﮭﺎ.

ﺗﻮﻗﻴﻊ ﺍﻟﺴﺎﺩﺓ ﺍﳌﺸﺮﻓﻮﻥ : ﺃﺳﺘﺎﺫ ﺩﻛﺘﻮﺭ /ﻳﺴﺮﻯ ﺻﺎﱀ : ﺃﺳﺘﺎﺫ ﺩﻛﺘﻮﺭ /ﻣﺮﻓﺖ ﻋﻠﻰ ﺍﺑﻮ ﺳﺘﻴﺖ

أ.د. ﺟــﻤــﺎل ﻓــﮭــﻤــﻰ ﺭﺋﻴﺲ ﳎﻠﺲ ﻗﺴﻢ ﺍﻟﻨﺒﺎﺕ ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ – ﺟﺎﻣﻌﺔ ﺍﻟﻘﺎﻫﺮﺓ

ﺍﻟﺘﻜﺴﲑ ﺍﳊﻴﻮﻯ ﻟﻠﻤﺮﻛﺒﺎﺕ ﺍﻻﺭﻭﻣﺎﺗﻴﺔ ﻣﺘﻌﺪﺩﺓ ﺍﳊﻠﻘﺎﺕ ﺍﳍﻴﺪﺭﻭﻛﺮﺑﻮﻧﻴﺔ ﰱ ﺯﻳﺖ ﺍﻟﺒﺘﺮﻭﻝ ﺍﳌﻠﻮﺛﻪ ﻟﻠﺒﻴﺌﺔ

ﺇﻋﺪﺍﺩ

ﻋﺒﲑ ﻣﻌﻮﺽ ﺑﺮﺗﻼ

ﺭﺳﺎﻟﺔ ﻣﻘﺪﻣﺔ ﺇﱄ ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ ﻛﺠﺰﺀ ﻣﻦ ﻣﺘﻄﻠﺒﺎﺕ ﺍﳊﺼﻮﻝ ﻋﻠﻲ ﺩﺭﺟﺔ ﺩﻛﺘﻮﺭﺍﺓ ﰱ ﻓﻠﺴﻔﺔ ﺍﻟﻌﻠﻮﻡ ( ﻣﻴﻜﺮﻭﺑﻴﻮﻟﻮﺟﻰ )

ﻗﺴﻢ ﺍﻟﻨﺒﺎﺕ ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ ﺟﺎﻣﻌﺔ ﺍﻟﻘﺎﻫﺮﺓ ( 2013 )