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Aerobic Microbial Degradation of Chloroaromatic Compounds Polluting the Environment

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Aerobic Microbial Degradation of Chloroaromatic Compounds Polluting the Environment Cairo University Faculty of Science Botany Department AEROBIC MICROBIAL DEGRADATION OF CHLOROAROMATIC COMPOUNDS POLLUTING THE ENVIRONMENT Presented By Ola Abdel A'al Abdel A'aty Khalil M.Sc., Banha University, 2005 A Thesis Submitted to Faculty of Science In Partial Fulfillment of the Requirements for the Degree of Ph.D. of Science (Microbiology) Botany Department Faculty of Science Cairo University (2011) Cairo Univ ersity Faculty of Science Botany Department AEROBIC MICROBIAL DEGRADATION OF CHLOROAROMATIC COMPOUNDS POLLUTING THE ENVIRONMENT Presented By Ola Abdel A'al Abdel A'aty Khalil M.Sc., Banha University, 2005 A Thesis Submitted to Faculty of Science In Partial Fulfillment of the Requirements for the Degree of Ph.D. of Science (Microbiology) Under Supervision of Prof. Prof. Youssry El-Sayed Saleh Mervat Aly Mohamed Abo-State Professor of Microbiology Professor of Microbiology Faculty of Science National Center for Research & Cairo University Radiation Technology Botany Department Faculty of Science Cairo University (2011) <<< <<<Üéu†Ö]<à·†Ö]<]<ÜŠe < َوَأ6َ8َْلَوَأ6َ8َْل اُ>اُ> Aَ>َْ?@َAََ@?َْ< اCDَFِGَْباCDَFِGَْب واHَIَFْJِGْواHَIَFْJِGْ َوI@KAَََ<َوMَGْMَG CLَCLَ >ََI@KAَْ Mَُ@PْOَMَُ@PْOَ ْNُFOَْNُFOَ َوَآCَنَوَآCَن SُTْUَSُTْUَ اِ>اِ> Aَ>َْ?@َAََ@?َْ< CIً?WِAَCIً?WِAَ < <<Üé¿ÃÖ]<]<Ñ‚‘ <<DMMOE<íè<íèaa ð^ŠßÖ]ð^ŠßÖ]<ì…ç‰<ì…ç‰ ABSTRACT Student Name: Ola Abdel A'al Abdel A'aty Khalil Title of the thesis: Aerobic Microbial Degradation of Chloroaromatic Compounds Polluting the Environment Degree: (Microbiology) Eight soil and sludge samples which have been polluted with petroleum wastes for more than 41 years were used for isolation of adapted indigenous microbial communities able to mineralize the chloroaromatic compounds [3-chlorobenzoic acid (3-CBA), 2,4-dichlorophenol (2,4-DCP), 2,6-dichlorophenol indol phenol (2,6-DCPP) and 1,2,4-trichlorobenzene (1,2,4-TCB)] and use them as a sole carbon and energy sources. From these communities, the most promising bacterial strain MAM-24 which has the ability to degrade the four chosen aromatic compounds was isolated and identified by comparative sequence analysis for its 16S-rRNA coding genes and it was identified as Bacillus mucilaginosus HQ 013329. Degradation percentage was quantified by HPLC. Degradation products were identified by GC-MS analysis which revealed that the isolated strain and its mutant dechlorinated the four chloroaromatic compounds in the first step forming acetophenone which is considered as the corner stone of the intermediate compounds. Keywords : Chloroaromatic compounds – Pollution – Bacteria Supervisors: Signature: 1- Prof. Dr. Youssry El Sayed Saleh 2- Prof. Dr. Mervat Aly Mohamed Abo-State Prof. Dr. Maimona Abdel-Aziz Kord Chairman of Botany Department Faculty of Science- Cairo University APROVAL SHEET FOR SUMISSION Thesis Title: Aerobic Microbial Degradation of Chloroaromatic Compounds Polluting the Environment Name of candidate: Ola Abdel A'al Abdel A'aty Khalil This thesis has been approved for submission by the supervisors: 1- Prof. Dr. Youssry El-Sayed Saleh Signature: 2- Prof. Dr. Mervat Aly Mohamed Abo-State Signature: Prof. Dr. Maimona Abdel-Aziz Kord Chairman of Botany Department Faculty of Science- Cairo University LIST OF ABBREVIATIONS Abbrev. Full Term 1,2,4-TCB 1,2,4-Trichlorobenzene 2,4-DCP 2,4-Dichlorophenol 3-CBA 3-chlorobenzoic acid BLAST Basic logical alignment search tool Blastn Somewhat similar sequences BOD Biochemical oxygen demand CB Chlorinated benzene CBz Chlorobenzoates CDB Chloroaromatic degrading bacteria CFU/ml Colony forming unit/ml Clustal W2 Program for comparing the aligned sequences COD Chemical oxygen demand EBI European biotechnology information GC-MS Gas chromatography/mass spectrometry HPLC High performance liquid chromatography KGy Kilo-gray NCBI National Center for Biotechnology Information PCBs Polychlorinated biphenyls (PCBs) PCP Pentachlorophenol RT Retention time TBC Total bacterial count TCA Tricarboxlic acid cycle TE buffer Tris-HCL buffer used to store DNA and RNA LIST OF TABLES Table No. Title Page No. Table (1): Sampling sites represented different sources of indigenous microbial communities. ................................73 Table (2): Growth & concentration of extracellular protein of different bacterial communities on 1 mM of 3-chlorobenzoic acid (3-CBA)..........................89 Table (3): Growth & concentration of extracellular protein of different bacterial communities on 1mM of 2,4-dichlorophenol (2,4-DCP).........................................92 Table (4): Growth & concentration of extracellular protein of different bacterial communities on 1mM of 2,6-dichlorophenol indol phenol (2,6-DCPP). ................94 Table (5): Growth & concentration of extracellular protein of different bacterial communities on 15µM of 1,2,4-trichlorobenzene (1,2,4-TCB). ...............................97 Table (6): Count after incubation period for 28 days of different mixed indigenous bacteria on different chloroaromatic compounds ...........................................106 Table (7): Count of different indigenous bacterial communities on different types of media. .....................110 Table (8): Determination of the ability of the indigenous isolated strains to grow on different chloroaromatic compounds at different concentrations.................................................................112 Table (9): Characterization of the most promising isolates. ..........113 Table (10): Growth, concentration of extracellular protein & concentration of Cl - of isolate MAM-24 on different concentrations of 3-chlorobenzoic acid (3-CBA).........................................................................115 Table (11): Growth, concentration of extracellular protein & concentration of Cl - of isolate MAM-27 on different concentrations of 3-chlorobenzoic acid (3-CBA).........................................................................119 LIST OF TABLES (Cont…) Table No. Title Page No. Table (12): Growth, concentration of extracellular protein & concentration of Cl - of isolate MAM-29 on different concentrations of 3-chlorobenzoic acid (3-CBA).........................................................................122 Table (13): Growth, concentration of extracellular protein & concentration of Cl - of isolate MAM-33 on different concentrations of 3-chlorobenzoic acid (3-CBA).........................................................................126 Table (14): Growth, concentration of extracellular protein & concentration of Cl - of isolate MAM-3 on different concentrations of 3-chlorobenzoic acid (3-CBA).........................................................................130 Table (15): Growth, concentration of extracellular protein & concentration of Cl - of E. cloaceae MAM-4 on different concentrations of 3-chlorobenzoic acid (3-CBA).........................................................................133 Table (16): Degradation percentage of 3-chlorobenzoic acid (3-CBA) after 21 days by HPLC...................................139 Table (17): Growth, concentration of extracellular protein & concentration of Cl - of isolate MAM-24 on different concentrations of 2,4-dichlorophenol (2,4-DCP). .....................................................................142 Table (18): Growth, concentration of extracellular protein & concentration of Cl - of isolate MAM-27 on different concentrations of 2,4-dichlorophenol (2,4-DCP). .....................................................................145 Table (19): Growth, concentration of extracellular protein & concentration of Cl - of isolate MAM-29 on different concentrations of 2,4-dichlorophenol (2,4-DCP). .....................................................................149 Table (20): Growth, concentration of extracellular protein & concentration of Cl - of isolate MAM-33 on different concentrations of 2,4-dichlorophenol (2,4-DCP). .....................................................................152 LIST OF TABLES (Cont…) Table No. Title Page No. Table (21): Growth, concentration of extracellular protein & concentration of Cl - of isolate MAM-3 on different concentrations of 2,4-dichlorophenol (2,4-DCP). .....................................................................155 Table (22): Growth, concentration of extracellular protein & concentration of Cl - of E. cloaceae MAM-4 on different concentrations of 2,4-dichlorophenol (2,4-DCP). .....................................................................159 Table (23): Count of different indigenous isolates on different concentrations of 2,4- dichlorophenol (2,4-DCP). .....................................................................162 Table (24): Degradation percentage of 2,4-dichlorophenol (2,4-DCP) after 21 days by HPLC. ...............................168 Table (25): Growth, concentration of extracellular protein & concentration of Cl –of isolate MAM-24 on different concentrations of 2,6-dichlorophenol indol phenol (2,6-DCPP)...............................................172 Table (26): Growth, concentration of extracellular protein & concentration of Cl - of isolate MAM-27 on different concentrations of 2,6-dichlorophenol indol phenol (2,6-DCPP)...............................................175 Table (27): Growth, concentration of extracellular protein & concentration of Cl - of isolate MAM-29 on different concentrations of 2,6-dichlorophenol indol phenol (2,6-DCPP)...............................................180 Table (28): Growth, concentration of extracellular protein
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