الفهرس 
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Abstract
The present study is concerned with three approaches;
The first approach was to isolate PAHs degrading bacteria from both oil contaminated soil and crude oil collected from different Egyptian locations, to screen the most efficient degraders and select them for completing degradation study. The molecular techniques such as, Polymerase Chain Reaction (PCR) amplification and sequencing of 16SrRNA genes as well as RAPD-PCR fingerprinting technique were used for identification and genetic differentiation at molecular level of the selected isolates. The relationship between PAHs removal and catabolic degradation genes (Alk, AlkB, NahAC, PAH-RHDα, C12O and C23O) were investigated.
The main results of these parts were summarized as the following:
• The total bacterial propagules were recovered on minimal basal salt (MBS) medium inverse proportion with the number of PAHs rings, where the total propagules recovered on MBS medium amended with naphthalene >phenanthrene> anthracene> pyrene.
• Forty two isolates were isolated from different Egyptian samples (crude oil and soil contaminated with crude oil) on different PAHs (twenty seven strains were gram negative and fifteen were gram positive) ,
• Four isolates (ASU-01, ASU-06, ASU-016, and ASU-035) out of 42, were selected based on difference in their shape, color of colony and high level of growth as the most efficient PAHs degraders.
• Based on both phenotypic and genotypic characterization, all selected isolates were gram negatives, short- rods, aerobic, non spore forming, non-acid fast, and belonged to Proteobacteria group. Isolates ASU-01, ASU-06, ASU-016 and ASU-035 were identified based on 16S rRNA gene sequencing and phylogenetic analysis as Enterobacter hormaechei (99 %), Sphingomonas koreensis (99 %), Pseudomonas pseudoalcaligenes (100 %), and Achromobacter denitrificans (97 %), respectively.
• All selected isolates were capable of producing indigo on minimal basal salt media amended with indole.
• All selected organisms revealed the expression of five PAHs catabolic genes (Alk, AlkB, NahAC, C12O and C23O) which responsible for PAHs degradation.
The second approach was to study the effect of PAHs on the growth parameters of the four selected organisms, to determine the biodegradation percentage along 15 days of incubation by HPLC; to investigate the biosutfactant production activities and percentages of cell surface hydrophobicty, and to detect specific activity of some PAHs degrading enzymes.
The results of this part were summarized as the following:
• The fastest growing strain was S. korineesis with maximum growth rate (0.01h-1), (0.006 h-1), (0.0084 h-1) (0.0060h-1) and (0.013h-1) when it was grown in liquid MBS medium amended with 100 mg/l of naphthalene, phenanthrene, anthracene, pyrene or mixed of the four PAHs, respectively.
• S. korineesis was the most efficient PAHs degrading bacteria where it could completely remove 100 mg/l of naphthalene, phenanthrene, anthracene and 92.7 % of pyrene during 15 days incubation.
• Mixed PAHs (naphthalene, phenanthrene, anthracene, pyrene) showed synergetic effect on HMW-PAH (pyrene) degradation with E. hormaechei, S. koreensis, while antagonistic effect revealed with P. pseudoalcaligenes and A. denitrificans.
• The ability of each isolates to produce biosurfactant depends on the solubility of carbon source. By decreasing the solubility of PAHs compound, the activity of biosurfactants increased: naphthalene < phenanthrene < anthracene < pyrene.
• Percentage of cell surface hydrophobicity was proportional with production of biosurfactants and subsequently the hydrophobicity of substrate.
• All selected isolates showed the ability to produce dioxygenase, catechol 1,2 dioxygenase and catechol 2,3 dioxygenase with variable activities.
• The activities of these enzymes were different according to the used substrate.
• The induction of catechol 1,2 dioxygenese was generally higher than catechol 2,3 dioxygenase. This indicated that the ortho cleavage pathway was more favorable than meta cleavage one for most of strains.
The third approach was to determine the metabolic products of S. koreensis through GC/MS analysis because it was the most efficient and rapidly degrading organisms and postulate its proposed pathways of two, three and four rings PAHs.
The results revealed the following:
• Total seven metabolic products were produced from naphthalene degradation along 6 to 15 days. 1-Methoxy naphthalene, 1-hydroxy-2-naphthoat, 2 hydroxy 4-methoxy cinnamate, salicylate, phthalate, phthalate 3,4-dihydrodiol, pyruvate.
• Four metabolites were produced from phenanthrene degradation, 1- hydroxyl phenanthrene, phthalate, dihydroxycis, cis muconate semialdehyde and 2-hydroxy 4methoxy cinnamate.
• During degradation of pyrene, Thirteen metabolites were detected 1-hydyoxy pyrene, 4,5 dihydroxy pyrene, phenanthrene-4,5-dicarboxylate, 3,4-dihydroxy phenanthrene, 1-hydroxy-2-naphthoate, benzocoumarin, trans-2-carboxy benzalpyruvate, phthalate, phthalate 3,4-dihydrodiol, dihydroxy phthalate, carboxy-cis,cis-muconate, 1-methoxy2-hydroxy pyrene and 1-methoxyphenanthrene .
• The main product of biodegradation pathway was phthalate .
The present study is concerned with three approaches;
The first approach was to isolate PAHs degrading bacteria from both oil contaminated soil and crude oil collected from different Egyptian locations, to screen the most efficient degraders and select them for completing degradation study. The molecular techniques such as, Polymerase Chain Reaction (PCR) amplification and sequencing of 16SrRNA genes as well as RAPD-PCR fingerprinting technique were used for identification and genetic differentiation at molecular level of the selected isolates. The relationship between PAHs removal and catabolic degradation genes (Alk, AlkB, NahAC, PAH-RHDα, C12O and C23O) were investigated.
The main results of these parts were summarized as the following:
• The total bacterial propagules were recovered on minimal basal salt (MBS) medium inverse proportion with the number of PAHs rings, where the total propagules recovered on MBS medium amended with naphthalene >phenanthrene> anthracene> pyrene.
• Forty two isolates were isolated from different Egyptian samples (crude oil and soil contaminated with crude oil) on different PAHs (twenty seven strains were gram negative and fifteen were gram positive) ,
• Four isolates (ASU-01, ASU-06, ASU-016, and ASU-035) out of 42, were selected based on difference in their shape, color of colony and high level of growth as the most efficient PAHs degraders.
• Based on both phenotypic and genotypic characterization, all selected isolates were gram negatives, short- rods, aerobic, non spore forming, non-acid fast, and belonged to Proteobacteria group. Isolates ASU-01, ASU-06, ASU-016 and ASU-035 were identified based on 16S rRNA gene sequencing and phylogenetic analysis as Enterobacter hormaechei (99 %), Sphingomonas koreensis (99 %), Pseudomonas pseudoalcaligenes (100 %), and Achromobacter denitrificans (97 %), respectively.
• All selected isolates were capable of producing indigo on minimal basal salt media amended with indole.
• All selected organisms revealed the expression of five PAHs catabolic genes (Alk, AlkB, NahAC, C12O and C23O) which responsible for PAHs degradation.
The second approach was to study the effect of PAHs on the growth parameters of the four selected organisms, to determine the biodegradation percentage along 15 days of incubation by HPLC; to investigate the biosutfactant production activities and percentages of cell surface hydrophobicty, and to detect specific activity of some PAHs degrading enzymes.
The results of this part were summarized as the following:
• The fastest growing strain was S. korineesis with maximum growth rate (0.01h-1), (0.006 h-1), (0.0084 h-1) (0.0060h-1) and (0.013h-1) when it was grown in liquid MBS medium amended with 100 mg/l of naphthalene, phenanthrene, anthracene, pyrene or mixed of the four PAHs, respectively.
• S. korineesis was the most efficient PAHs degrading bacteria where it could completely remove 100 mg/l of naphthalene, phenanthrene, anthracene and 92.7 % of pyrene during 15 days incubation.
• Mixed PAHs (naphthalene, phenanthrene, anthracene, pyrene) showed synergetic effect on HMW-PAH (pyrene) degradation with E. hormaechei, S. koreensis, while antagonistic effect revealed with P. pseudoalcaligenes and A. denitrificans.
• The ability of each isolates to produce biosurfactant depends on the solubility of carbon source. By decreasing the solubility of PAHs compound, the activity of biosurfactants increased: naphthalene < phenanthrene < anthracene < pyrene.
• Percentage of cell surface hydrophobicity was proportional with production of biosurfactants and subsequently the hydrophobicity of substrate.
• All selected isolates showed the ability to produce dioxygenase, catechol 1,2 dioxygenase and catechol 2,3 dioxygenase with variable activities.
• The activities of these enzymes were different according to the used substrate.
• The induction of catechol 1,2 dioxygenese was generally higher than catechol 2,3 dioxygenase. This indicated that the ortho cleavage pathway was more favorable than meta cleavage one for most of strains.
The third approach was to determine the metabolic products of S. koreensis through GC/MS analysis because it was the most efficient and rapidly degrading organisms and postulate its proposed pathways of two, three and four rings PAHs.
The results revealed the following:
• Total seven metabolic products were produced from naphthalene degradation along 6 to 15 days. 1-Methoxy naphthalene, 1-hydroxy-2-naphthoat, 2 hydroxy 4-methoxy cinnamate, salicylate, phthalate, phthalate 3,4-dihydrodiol, pyruvate.
• Four metabolites were produced from phenanthrene degradation, 1- hydroxyl phenanthrene, phthalate, dihydroxycis, cis muconate semialdehyde and 2-hydroxy 4methoxy cinnamate.
• During degradation of pyrene, Thirteen metabolites were detected 1-hydyoxy pyrene, 4,5 dihydroxy pyrene, phenanthrene-4,5-dicarboxylate, 3,4-dihydroxy phenanthrene, 1-hydroxy-2-naphthoate, benzocoumarin, trans-2-carboxy benzalpyruvate, phthalate, phthalate 3,4-dihydrodiol, dihydroxy phthalate, carboxy-cis,cis-muconate, 1-methoxy2-hydroxy pyrene and 1-methoxyphenanthrene.
•The main product of biodegradation pathway was phthalate.