الفهرس 
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Abstract
Water polluted by agricultural wastes is caused by the excessive use of pesticides in many agricultural areas and results in underground water contamination. Synthetic pesticides are considered a threat in many agricultural areas.
Biodegradation is an ecofriendly way of converting pesticides into simple nontoxic compounds by means of enzymes generally produced by microbes.
This study was conducted to isolate, identify the potent bacterial isolates capable of degrading chlorpyrifos, malathion and azoxystrobin pesticides, detect pesticides’ degrading genes and their expression level, optimize some cultural conditions to achieve maximum chlorpyrifos degradation to identify the metabolites and the probable chlorpyrifos degradation pathway.
To achieve these objectives twenty bacterial isolates were recovered from water samples collected from agricultural drains located in three governorates including Al Qalyubia, Al Dakahlia and Al fayoum.
Fourteen out of the twenty bacterial isolates were able to degrade chlorpyrifos, malathion and azoxystrobin separately at different concentrations (100 – 700 mgl-1).
Based on the morphological, cultural, biochemical and molecular characteristics the fourteen bacterial isolates were identified as Klebsiella sp. QBM11, Enterobacter cloacae QBM12, Raoultella planticola FYF35, Bacillus licheniformis FYZ33, Kosakonia sp. FYF33, Enterobacter cloacae QBM13, Klebsiella aerogenes QBF13, Klebsiella sp. QBZ11, Raoultella ornithinolytica DSM23, Raoultella ornithinolytica DSM24, Klebsiella variicola DSM25, Klebsiella pneumonia DSF23, Bacillus sp. FYM31 and Klebsiella pneumonia QBF11 and deposited into GenBank with the accession numbers starting from OK325585 to OK325598 respectively.
Metallo-hydrolase enzyme encoded by oph gene was detected in three bacterial strains, Klebsiella aerogenes QBF13, Kosakonia sp. FYF33 and Bacillus sp. FYM31 and deposited into GenBank with the accession numbers ON033836, OM502410 and OM502411 respectively. While aminotransferase class III-fold pyridoxal phosphate-dependent (PLP) enzyme encoded by phn gene was detected in Klebsiella sp. QBM11, K. variicola DSM25 and E. cloacae QBM12 strains and deposited into GenBank with the accession numbers OM502408, ON033835 and OM502409 respectively.
HPLC analysis of chlorpyrifos residues revealed that E. cloacae QBM12, K. variicola DSM25, K. aerogenes QBF13, Kosakonia sp. FYF33, Klebsiella sp. QBM11 and Bacillus sp. FYM31 degraded 100 & 100, 100 & 100, 99.7 & 100, 94.6 & 100, 91.6 & 100 and 83.8 & 86.8 % after 9 and 12 days of incubation respectively. While HPLC analysis of malathion residues revealed that E. cloacae QBM12, K. variicola DSM25, K. aerogenes QBF13, Kosakonia sp. FYF33, Klebsiella sp. QBM11 and Bacillus sp. FYM31 degraded 73.1 & 75.3, 75.3 & 78.1, 73.4 & 76.2, 70 & 73.4, 66.5 & 69.6 and 66.5 & 70.1 % after 9 and 12 days of incubation respectively.
HPLC analysis of azoxystrobin residues revealed that E. cloacae QBM12, K. variicola DSM25, K. aerogenes QBF13, Kosakonia sp. FYF33, Klebsiella sp. QBM11 and Bacillus sp. FYM31 degraded 27.9 & 30.7, 42.5 & 58.9, 30.1 & 34.1, 32.4 & 39.5, 25.8 & 27.7 and 29.4 & 30.1 % after 9 and 12 days of incubation respectively. HPLC analysis of chlorpyrifos, malathion and azoxystrobin residues revealed that E. cloacae QBM12, K. variicola DSM25, K. aerogenes QBF13, Kosakonia sp. FYF33, Klebsiella sp. QBM11 and Bacillus sp. FYM31 were able to degrade 100, 75.3 & 30.7, 100, 78.1 & 58.9, 100, 76.2 & 34.1, 100, 73.4 & 39.5, 100, 69.6 & 27.7 and 86.8, 70.1 & 30.1 % respectively after 12 days of incubation.
Quantitative real time PCR analysis revealed that the expression level of oph gene in K. aerogenes QBF13 and Kosakonia sp. and phn gene in K. variicola DSM25 treated with chlorpyrifos and azoxystrobin pesticides separately showed significant increase in their expression levels for chlorpyrifos treatment by higher expression level than azoxystrobin treatment for all the selected strains.
As Kosakonia sp. FYF33 is the safest bacterial isolate among the tested pesticides degraders, it was selected for further experiments; optimization of some cultural conditions for maximum chlorpyrifos degradation and elucidation of chlorpyrifos degradation pathway.
To achieve maximum chlorpyrifos biodegradation by Kosakonia sp. FYF33, the effect of temperature, pH and incubation time were evaluated using Plackett-Burman Design and Central Composite Design. Maximum chlorpyrifos degradation (96.1%) was achieved at optimum conditions, pH 8 and 5 days of incubation. Based on GC-MS analysis, a probable degradation pathway for chlorpyrifos degradation by Kosakonia sp. FYF33 has been proposed, that followed the pathway from chlorpyrifos to 3, 5, 6-trichloro-2-pyridinol (TCP) to 2-Ethyl-6-methyl-3-hydroxypyridine and then entered the tricarboxylic acid (TCA) cycle.