I= STUDIES OF COPPER AND CHROMIUM RESISTANCE AND THEIR REMEDIATION BY INDIGENOUS BACTERIAL STRAINS
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Title of Thesis
STUDIES OF COPPER AND CHROMIUM RESISTANCE AND THEIR REMEDIATION BY INDIGENOUS BACTERIAL STRAINS
Author(s)
Uzma Badar
Institute/University/Department Details
Department of Genetics/ University of Karachi
Session
2003
Subject
Genetics
Number of Pages
198
Keywords (Extracted from title, table of contents and abstract of thesis)
copper resistance, chromium resistance, bacterial strains, plasmid dna, cmg457, cmg462, cmg463, cmg458

Abstract
Several bacterial strains were screened from CMG stock culture on the basis of multiple metal resistance characters. The others were isolated from metal contaminated sites of Karachi. Among the isolated ones only three bacterial strains were selected, one isolated from Karachi tannery effluents sector 7-A Korangi industrial area and two from foundry soil of Karachi Shipyard and Engineering Works Ltd. The selection criteria were multiple metal resistance and high resistance against copper and chromium so that these strains could be exploited in future for remediation purpose. These isolated bacterial strains were designated as CMG458. CMG462 and CMG463 and identified by 16S rRNA gene sequencing. The bacterial strain CMG458 exhibited closed homology with Pseudomonas sp and CMG462 and CMG463 with Pseudomonas stutzeri. One strain, CMG457 was selected from CMG stock for further studies, this was identified as Enterobacter sp. by I6S rRNA gene sequencing. Among all the strains CMG462 and CMG463 showed highest resistance against copper i.e. 8mM and l0mM respectively whereas CMG458 and CMG457 also showed high resistance but relatively low i.e., 5 and 4 mM respectively. Further CMG462 and CMG463 also showed resistance against hexavalent chromium [Cr(Vl)] upto 1 mM and reduced Cr(VI) anaerobically from a 100 μM Cr(VI) solution. The highest removal of chromium was by CMG463 i .e.88:4μmol/l (88% of that supplied; specific rate was 3.0 nmol/mg of protein/h), while 58.3μmol/1 (58%) were removed by CMG462. These strains were also compared with the strains isolated from an uncontaminated coastal site in the UK. Strain K3 was Cr-sensitive, partially lysed with Cr(VI), but had the highest removal of chromate a aerobically i .c. 92.1 μ mol/l (92%of that supplied) a t a specific rate of 71.5 n mol/mg of protein/h. Analysis of cell sections using transmission electron microscopy with energy dispersive x-ray analysis (EDAX) showed intracellular chromium in Pseudomonas stutzeri but the Bacillus sp. precipitated chrome extracellularly. The strains from the Cr-contaminated sites did not remove more Cr(VI), overall, than Cr-unstressed bacteria. Only CMG462 and CMG463 accumulated copper at a high concentration from the solution i.e. 1236.97 μ mol/mg of protein/h. Analysis of transmission electron microscopy and energy dispersive x-ray analysis revealed presence of copper intracellular and extracellular both. Stra5ns i.e. CMG462, CMG463, and CMG458 were found to carry single high molecular weight plasmid DNA while two plasmids were present in CMG457; they were designated as pCMG462, pCMG463, pCMG458, plCMG457 (~20 kb) and p2CMG457 (~2.0kb) ,respectively. The copper resistance in CMG457 was shown to be mediated by one plasmid (pICMG457), this was confirmed by successful transformation and curing ofp1CMG457. A complete peo operon (pcoABCDRSE) in CMG457 was identified by PCR and pcoACD gene sequences showed closed homology with the pcoACD gene of E. coli plasmid pRJI004. Whereas in CMG462, CMG463 and CMG458, copper resistance might be plasmid mediated, plasmids were unable to be transformed and cured but copA gene was identified by PCR, copA sequence of CMG463 showed -100% homology with copA gene of P. syringae plasmid pT23D while low homology with CMG458. This was considered to have a novel copper resistance copA like gene, and it has no restriction sites like copA sequence of P syringae plasmid pPT23D, it was cloned in pGEM-t easy vector and transformed into JMl09 where it was expressed successfully. While whole cop operon (copABCDRS) could not be identified.

As CMG463 removed highest concentration of copper it was also exploited for the removal of copper by developing bioreactor/biofilters and copper level in the out flow of bioreactor determined the removal efficiency. CMG463 cells were immobilized by developing biofilm on sponge; it was also examined by scanning electron microscopy. The biofilter demonstrated efficient removal of copper i.e. 90% from solution containing copper. To conclude CMG463 having copA gene for copper resistance and showing highest removal of copper and maximum reduction of chromate could be exploited for remediation of sites contaminated with copper and chromium.
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1966.08 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
213.46 KB
2 1 Literature 4
266.68 KB
  1.1 Occurrence Of Heavy Metal In Effluents 4
  1.2 Chromium 10
  1.3 Toxicity Of Heavy Metal 11
  1.4 Bacterial Resistance To Heavy Metals 15
  1.5 Microbial Biotechnology For The Remediation 26
3 2 Materials And Methods 31
145.7 KB
  2.1 Growth Media And Chemicals 31
  2.2 Sample Collection Enrichment, Isolation And Purification Of Bacterial Strains 31
  2.3 Preservation Of Bacterial Culture 31
  2.4 Maximum Tolerable Concentration ( Mtc ) Of Heavy Metal 33
  2.5 Maximum Tolerable Concernt4ration( Mtc ) Of Antibiotics 33
  2.6 Isolation Of Plasmid Dna 33
  2.7 Curing Of Plasmid Dna 34
  2.8 Isolation Of Genomic Dna 34
  2.9 Transformation Of Plasmid Dna 37
  2.10 Agarose Gel Electrophoresis 35
  2.11 Reduction Of Hexavalent Chromium( Cr Vi) Into Trivalent Chromium(Cr Iii) 35
  2.12 Chromate (Cr Vi ) Assay 36
  2.13 Protein Assay 36
  2.14 Accumulation Of Copper 36
  2.15 Copper Assay 37
  2.16 Electron Microscopy 37
  2.17 Polymerase Chain Reaction( Pcr ) 43
  2.18 Alkali Blotting 44
  2.19 Restriction Fragment Length Polymorphisms( Rflps ) 44
  2.20 Cloning Of Novel Copper Resistance Gene In Pgem T-Easy Vector 44
  2.21 Purification And Sequencing Of Pcr Products 46
  2.22 Bioreactors/ Biofilters For The Removal Of Copper 47
4 3 Results 49
152.77 KB
  3.1 Isolation, Selection And Characterization Of Bacterial Strains 49
  3.2 Selection Of Bacterial Strains Form CMG Culture Stock 49
  3.3 Sample Collection, Enrichment, Isolation And Purification Of Bacterial Strains 49
  3.4 Identification Of Bacterial Strains 49
  3.5 Maximum Tolerable Concentration Of Heavy Metal Salts 50
  3.6 Maximum Tolerable Of Antibiotics 50
  3.7 Isolation Of Plasmid Dna 50
  3.8 Curing Of Plasmid Dna 51
  3.9 Transformation Of Plasmid Dna 51
  3.10 Conclusion 51
5 4 Chromium And Copper 65
423.38 KB
  4.1 Maximum Tolerable Concentration And Reduction Of Chromate 65
  4.2 Electron Microscopy 66
  4.3 Reduction/ Removal Of Chromate By Resting Cells 67
  4.4 Maximum Tolerable Concentration Of Copper 68
  4.5 Accumulation/Removal Of Copper 68
  4.6 Bios Sorption Of Copper 69
  4.7 Conclusions 69
6 5 Molecular Genetics Of Copper Resistance 96
463.1 KB
  5.1 Screening Of copA/pcoA Gene By Polymerase Chain Reaction( Pcr ) 96
  5.2 Restriction Fragment Length Polymorphisms( Rflps ) Of Pcr Product Of Copa/Pcoa Gene 96
  5.3 Expand Long Template PCR Of cop And Pco Operon 96
  5.4 Alkali/ Southern Blotting 97
  5.5 Screening Of Pco /Cop Operon In CMG457 With Intragenic Primers Of Each Gene 97
  5.6 Screening Of pco /cop Operon In CMG457 With Intergenic Primers 97
  5.7 Screening Of pco /cop Operon In CMG462, CMG463 And CMG458 With Intragenic Primers Of Each Gene 98
  5.8 Cloning Of Copper Resistance Gene Of CMG458 In Pgem -T Easy Vector 99
7 6 Removal Of Copper Via Bioreactor 100
185.9 KB
  6.1 Selection Of Support Materials 154
  6.2 Development Of Biofilm In Batch Culture 154
  6.3 Development Of Biofilm In Chemostat Culture 154
  6.4 Removal Of Copper From Lab-Scale Bioreactor 155
  6.5 Edax Of Biofilm Coated Sponge 155
  6.6 Standard Curve Of Copper Assay 155
8 7 Discussion And Conclusions 163
380.8 KB
  7.1 Bibliography 174
  7.2 Appendix A-D 194