I= GENETIC DIVERSITY AMONG BACTERIAL FLORA OF BALUCHISTAN COAST
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Title of Thesis
GENETIC DIVERSITY AMONG BACTERIAL FLORA OF BALUCHISTAN COAST

Author(s)
BUSHRA UZAIR
Institute/University/Department Details
Department of Microbiology/ University of Karachi
Session
2007
Subject
Microbiology
Number of Pages
289
Keywords (Extracted from title, table of contents and abstract of thesis)
genetic diversity, bacterial flora, baluchistan coast, phytate hydrolysing, zafrin, bushrin, s. aureus, b. subtilis

Abstract
The oceans offer abundant resources for research and development yet the potential of this domain as the basis for new biotechnologies remains largely unexplored. Study of marine microbial biodiversity is of vital importance for the understanding of the different processes of the ocean, which may present potent novel microorganisms for screening of bioactive compounds. As the microbial communities have a complex ecosystem processes, biodiversity study explore the distribution and roles in the habitat, estimation of microbial diversity is required for understanding the biogeography, community assembly and ecological processes.

To investigate the biodiversity of bacteria of Baluchistan coast, a total of 100 attached and free-living bacteria were isolated. Bacterial strains were identified by studying Gram reaction, cellular and colonial morphology biochemical reactions and. by QTS-24 (quick test strip) and confirmation of three selected strains was carried out by 16S rRNA gene sequence analysis. Out of the 100 bacterial isolates 81 were identified as Gram-negative and 19 were Gram positive. A remarkable percentage of isolated strains showed resistance against antibiotics and metals.

All of the 100 bacterial strains were analyzed for their ability to hydrolyze calcium phytate. Fifteen percent of the strains hydrolyzed calcium phytate on solid media and four strains showed hydrolysis of calcium phytate both on solid and liquid media. The strains which showed release of soluble phosphate as a result of hydrolysis of calcium phytate were selected for bioremediation of lanthanum nitrate from aqueous solution. A bioreactor was constructed using biofilm of mix culture of phytate hydrolysing strains (CMGI00l and CMGI083). Maximum removal of lanthanum was detected after 24 hrs of contact and white precipitates of lanthanum phosphate were observed on polyutherane foam cubes. These precipitates of lanthanum phosphate were observed using scanning electron microscope.

Isolated bacterial strains were checked for their ability to solubilize complex form of compounds containing phosphorous, four percent of the strains solubilized insoluble compounds both on solid and liquid media. The ability of the most efficient phosphate solubilizing bacterial strains to enhance the growth of maize plants was studied in potted soil experiment in green house. Experiments have showed that the strains, which released free phosphate from insoluble phosphate

Compounds, stimulated growth of maize plants. Isolated bacterial strains were also tested for their ability to produce antibacterial metabolite. A remarkable fraction 5 % of the strains exhibited antagonistic properties against antibiotic resistant clinical isolates and against indigenous marine bacteria. CMG1030 identified as Pseudomonas stutzeri exhibited a broad antibacterial spectrum.

To determine nature of antibacterial activity, different experiments were conducted. The antibacterial compound produced by CMG1030 was an extra cellular compound. Persistence of antibacterial activity in the presence of protease enzyme showed that antibacterial activity was not protein in nature. Siderophore involvement was not possible as antibacterial activity persisted at elevated concentration of Fe3+ ions in the growth medium. Screening of extra cellular chromosomal DNA showed that this strain does not harbor any plasmid DNA. To determine chemical nature of the antibacterial compound, extraction with organic solvents was done. Crude extract were prepared using organic solvents. Three novel antibacterial compounds designated as bushrin, zafrin and compound-1 were purified by column chromatography from the crude extract of CMGI030 and their structures were elucidated by spectroscopic techniques. The antibacterial compound bushrin was determined to be 7-(3-furyl)-3, 7-dimethyl 7, 8-dihydro-l-naphthalenol. The molecular formula C16H1602 of bushrin was deduced from HR-EIMS at m/z =240.2712

The antibacterial compound zafrin was determined to be 4b-methyl - 5, 6, 7, 8 tetrahydro-l (4b-H)-phenanthrenone. The molecular formula, C15H16O of zafrin was deduced from HR-EIMS at m/ z = 212.2871.

The antibacterial compound, compound-l was determined to be I-methyl-I, 4 dihydroquinoline. The molecular weight of compound-l was 145.2011 with a molecular formula C10 H11 N deduced from HR-EIMS.

The time kill experiment showed that bushrin was able to decrease the counts of S. nureus (ATCC33591) at MIC 60 ug/ml. The inhibitory activity was observed after at least. 8 hrs of exposure, whereas zafrin decreased the number of viable bacterial cells after 10 hr of exposure at MIC (75 ug/ml). A decrease in bacterial cell counts was more readily found when the bacterial cells were exposed to bushrin and zafrin at higher concentration.

The morphological changes in S. aureus (ATCC33591) and B. subtilis induced by bushrin were observed under phase contrast microscope and time lapse images were captured, decrease in size of cells of B. subtilis was observe after one minute of treatment with bushrin. When cells of S. aureus were treated with this compound complete lysis was observed immediately after treatment. Microscopic examination of exponentially growing cells of S. aureus treated with sub lethal concentration of bushrin suggests cell lysis and some ghost cells and big cells were also observed.

The morphological changes in S. aureus (ATCC33591) and B. subtilis induced by zafrin were also observed under phase contrast microscope. Two morphological changes were observed one was decrease in size of cells of B. subtilis immediately after treatment with zafrin and the other was the accumulation of large electron dense mass in the cytoplasm. When cells of S. aureus were treated sub lethal concentration of zafrin some very large cells were observed.

In conclusion coastal zone of Baluchistan was found to be rich source of bacterial flora which exhibited diverse characters.

Download Full Thesis
3334.64 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
338.52 KB
2 1 Baluchistan Coast Quantitative and Qualitative Analysis of Bacterial Flora 1
609.59 KB
  1.1 Introduction 1
  1.2 Material and Methods 11
  1.3 Results 17
  1.4 Discussion 49
3 2 Applied Bioremediation Construction of a bioreactor removing Lanthanum Nitrate from Aqueous Solution 58
542.89 KB
  2.1 Introduction 58
  2.2 Material and Methods 68
  2.3 Results 72
  2.4 Discussions 92
4 3 Screening of Bacterial Strains Soubilizing Insoluble Compounds and Their Potential as Plant Growth Promotion 97
563.48 KB
  3.1 Introduction 97
  3.2 Material and Methods 110
  3.3 Results 114
  3.4 Discussion 138
5 4 Isolation , Purification, and Structure Elucidation of Antibacterial Compounds from Bacteria of Baluchistan Coast 145
1218.17 KB
  4.1 Introduction 145
  4.2 Material and Methods 159
  4.3 Results 168
6 5 Discussion 239
418.49 KB
  5.1 Conclusion and Findings of the Study 250
  5.2 Publications 253
  5.3 References 254
  5.4 Appendix 283
  5.5 Abbreviation 289