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Title of Thesis

Zulfiqar Ali Saqib
Institute/University/Department Details
Department of Zoology/ University of the Punjab
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
insecticide, bacteria, insecticide disappearance, plasmids, insecticide degrading bacteria,

The soil samples were collected from agricultural fields with 5-10 years history of insecticidal spray from different parts of Punjab, Pakistan. Fifty nine bacterial strains resistant to organochlorine insecticides, i.e. endosulfan and heptachlor (cyclodiene group) were isolated. They were also capable of utilizing organophosphates (Anthio, Ethion, Quinalphos), pyrethroids (Bifenthrin, Cypermethrin) and a carbamate (Carbosulfan) as a carbon source in an inorganic M-9 agar medium. Thirty five of these bacterial strains were isolated from Lahore region and twenty four from Multan region. The minimum inhibitory concentration (MIC) of endosulfan and heptachlor ranged from 2 to 10 mg/ml, while that of organophosphates (OP€™s) from 1 to 6 mg/ml, of pyrethroids from 2 to 12 mg/ml and of carbosulfan from 1 to 4 mg/ml. On the basis of high MICs twenty four bacterial strains were finally selected for further analysis.

The optimum pH and temperature of the selected bacterial isolates was 6.5-7.5 and 30-42°C, respectively. The growth patterns of the isolates were studied in different media viz., LB, M9+glucose (50 mg/100 ml), M9+endosulfan (50 mg/100 ml) and M9+heptachlor (50 mg/100ml). In LB medium typical growth patterns were observed as compared with those in M9, in which prolonged lag or stationary phases were observed. Eight isolates, based upon the various biochemical tests, were identified as members of the genus Bacillus, two each of the genus Kurthia and Caryophanon and one each of Sporolactobacillus, Derxia, Streptococcus, Aureobacterium, Alcaligenes, Terrabacter, Rizhobacter, Azomonas and Pimelobacter.

They were also tested for sensitivity to eight heavy metals. The MICs of Cd2+ varied from 300-500 μg/ml, of CO2+ 1 00-200 μg/ml, of Cr6+ 300-500 μg/ml, of Cu2+ 350-450 μg/ml, of Hg2+ 1 00-200 μg/ml,. of Ni2+ 300-550 μg/ml, of Pb2+ 1500-2500 μg/ml and of Zn2+ 300-500 μg/ml.. The antibiotic sensitivity of isolates was also checked against nine antibiotics. The isolates were resistant to ampicillin, amoxycillin, cefixime and cefotoxim but were sensitive to minocyclin. Most of the bacteria were sensitive to cefazoline except for CMBLI 62, 75, 76 and 87, to chloramphenicol except for CMBLI 43, 44, 46, 53, 55 and 65 and tetracycline except for CMBLI 38, 53 and 55.

All isolates were found to harbour plasmids of 23 Kb except for CMBLI 62, which had five plasmids of 23, 13, 6.5, 4.2 and 2.9 Kb size. All the isolates could be cured of their plasmids, which made them sensitive to insecticides mixed in the growth media. The competent cells of E. coli C600 were successfully transformed with the plasmids of isolates. The transformed E. coli could grow on M9 selective medium containing 50 mg/100 ml of endosulfan/heptachlor. The 23 Kb plasmid was later successfully recovered from the transformants. It was concluded that the gene for insecticide degradation was located on the plasmids.

The total proteins of some selected isolates grown in LB, M9+glucose, M9+endosulfan and M9+heptachlor (50 mg/100 ml) were extracted in the sample buffer and were analyzed by 12% 8DS-PAGE. More protein bands were seen in isolates grown in LB media as compared with those in M9 media. Despite the suppression of certain protein bands in isolates grown in insecticide-containing media, protein bands of 64, 62, 59,50 and 37 Kd size were present in M9 as well as in control media. The presence of protein bands in insecticide stress media implicate their role in detoxification of insecticides. The isolated bacterial strains can be employed in the microbe based bioremediation of insecticide-contaminated soil and waste water

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
7350.8 KB
2 1 Introduction 1
665.68 KB
  1.1 Insecticides 2
  1.2 Pesticide Cycling In The Environment 4
  1.3 Factors Involved In Insecticide Disappearance 6
  1.4 Biotransformation 7
  1.5 Photochemical Mechanisms 10
  1.6 Physical Mechanisms 10
  1.7 Chemical Mechanisms 12
  1.8 Microbial Degradation Of Insecticides 13
  1.9 Bioremediation And Its Genetic Basis 16
  1.10 Aims And Objectives 32
3 2 Materials And Methods 35
661.13 KB
  2.1 Sample Collection 35
  2.2 Media Used 35
  2.3 Sterilization Of Media, Solutions And Apparatus 37
  2.4 Insecticides Used 37
  2.5 Insecticides Mixed Media 42
  2.6 Isolation Of Insecticide-Resistant Bacteria 42
  2.7 Purification Of Isolated Strains 43
  2.8 Physical And Biochemical Characterization Of Isolates 43
  2.9 Growth Curves Of Isolates 51
  2.10 Determination Of Optimum Growth Conditions 51
  2.11 Determination Of Optimum Temperature 51
  2.12 Determination Of Optimum Ph 52
  2.13 Antibiotic Resistance Of Isolates 52
  2.14 Molecular Analysis Of Insecticide Resistance Genes 53
  2.15 Transformation 55
  2.16 Sds-Page Analysis Of Total Proteins 56
4 3 Results 60
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  3.1 Insecticide Degrading Bacteria 60
  3.2 Cyclodiene Insecticide Resistant Isolates 60
  3.3 Effect Of Insecticide Concentrations On The Appearance Of Visible Colonies On Agar Plates 63
  3.4 Bacterial Isolates Selected For Further Study From Multan Region 66
  3.5 Bacterial Isolates Selected For Further Study From Lahore Region 71
  3.6 Resistance Of Insecticide Resistant Bacteria To Other Insecticides 71
  3.7 Morphological And Physical Characteristics Of The Isolates 76
  3.8 Biochemical Characteristics Of The Isolates 77
  3.9 Growth Curves Of Cyclodiene Insecticide Resistant Bacterial Isolates 91
  3.10 Effect Of Temperature On Growth Of Selected Isolates 102
  3.11 Effect Of Ph On Growth Of Selected Isolates 102
  3.12 Resistance Of Insecticide Resistant Bacteria To Heavy Metals 109
  3.13 Resistance Of Insecticide Resistant Bacteria Antibiotics 112
  3.14 Genetic Analysis Of The Selected Bacterial Isolates 113
  3.15 Sds-Page Pattern Of Total Proteins Of Insecticide Resistant Bacterial Isolates 128
5 4 Discussion 145
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  4.1 Isolation And Mic€™s Of The Insecticide Resistant Isolates 149
  4.2 Insecticide Resistant Isolates 151
  4.3 Growth Of Insecticide Resistant Bacterial Strains 154
  4.4 Diversity Of Insecticide Resistant Bacteria 157
  4.5 Growth Curves 162
  4.6 Effect Of Temperature And Ph On Bacterial Growth 163
  4.7 Heavy Metal Resistance Of Insecticide Resistant Bacteria 165
  4.8 Antibiotic Sensitivity Or Resistance Of Selected Bacterial Isolates 167
  4.9 Role Of Plasmid In Degradation Of Insecticides 168
  4.10 Curing Of Plasmids 171
  4.11 Sds-Page Of Total Proteins Of Isolates 171
  4.12 Strategies Of Exploiting The Isolates 171
6 5 References 179
833.78 KB