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Characterization of Bacterial Isolates from Kashmir Cave, Pakistan, and Their Potential Applications

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Characterization of Bacterial Isolates from Kashmir Cave, Pakistan, and Their Potential Applications Characterization of Bacterial isolates from Kashmir cave, Pakistan, and their Potential Applications. By Sahib Zada Department of Microbiology Quaid-i-Azam University Islamabad, Pakistan 2017 Characterization of Bacterial isolates from Kashmir cave, Pakistan, and their Potential Applications. A thesis Submitted in the Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY IN MICROBIOLOGY By Sahib Zada Department of Microbiology Quaid-i-Azam University Islamabad, Pakistan 2017 DECLARATION The material contained in this thesis is my original work and I have not presented any part of this thesis/work elsewhere for any other degree. Sahib Zada DEDICATED TO My Ammi, Abbu and Late Talha khan CONTENTS S. No. Title Page No. 1. List of Abbreviations i 2. List of Tables ii 3. List of Figures iii 4. Acknowledgements vi 5. Summary viii 6. Chapter 1: Introduction 1 7. Chapter 2: Review of Literature 18 8. Chapter 3: Paper 1 60 Paper 2 99 Paper 3 124 Paper 4 147 9. Chapter 5: Mn oxidation by Cavernicoles a. Abstract 170 b. Introduction 171 c. Methodology 180 d. Results 185 e. Discussion 201 f. References 204 10. Conclusions 206 11. Future Prospects 207 List of Abbreviations A Adenosine ATP Adenosine-5’-triphosphate BLAST Basic Local Alignment Search Tool BLASTX BLAST search using a translated nucleotide query °C Degree celsius Ca Calcium DNA Deoxyribonucleic acid e.g. Exempli gratia, for example et al. et alii/alia, and others Fe Iron Fig. Figure FTIR Fourier Transform Infra-Red spectroscopy HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid LBB Leucoberbelin Blue LDPE Low density polyethylene MCO Multi Copper Oxidase MEGAN 5 MEtaGenome ANalyzer Mn Manganese MOB Manganese Oxidizing Bacteria NaCl Sodium Chloride NCBI National Center for Biotechnology Information OD Optical Density PCR Polymerase Chain Reaction pH Power of Hydrogen RDP Ribosomal Database Project rRNA Ribosomal ribonucleic acid SEM Scanning Electron Microscopy SOD Superoxide Dismutase TBE Tris base, boric acid EDTA UV-Vis Ultra Violet Visible VOC Volatile Organic Compounds X-RD X-ray Powder Diffraction Zn Zinc List of Tables No. Title Page No. 1 List of largest and most studied caves world wide 9 2 List of caves in Pakistan 10 3 Cave microbes isolated from different caves in different country 13 through out the world 4 Different caves are studied for microbial diversity in different 37 countries 5 List of minerals obtained from smast-5 floor and smast-7 wall of 72 Kashmir smast samples 6 Concentration of elements in sample collected from cave floor and 72 outside cave soil (control) 7 Viable Cell Count of bacterial consortium in different media 109 compositions incubated at 37°C. 8 Mn(II) oxidizing Bacteria Isolated from Kashmir cave soil and 186 speleothem. List of Figure S.No. Page Title No. 1 Different types of caves and its formation including dissolution and 4 weathering. 2 Solutional cave formation 5 3 Limestone cave formation 5 4 Distribution of major groups of microbial communities in cave 12 environments by 16S rRNA gene sequencing 5 The process of calcium carbonate precipitation by bacteria 30 6 Bacteria serving as nucleation site for CaCO3 precipitation in the 31 sand particle 7 Distribution of major groups of microbial communities in cave 36 environments by 16S rRNA gene sequencing 8 Kashmir cave (smast), Nanseer Buner, Khyber Pakhtunkhwa, 65 Pakistan. White arrows show location of the cave, black arrow 105 shows entrance to the cave. 9 XRD patterns of Kashmir smast (a) from the floor and (b) from the 71 wall along-with the matched peaks of the mineral ICSD (Inorganic Crystal Structure Database) 10 Quantitative analysis of minerals, A. Wall soil sample smast-7, B. 73 Floor soil sample smast-5 11 Infrared spectra of Smast-7 wall 73 12 TGA (Thermogravimetric Analysis) plots of Kashmir smast 74 (sample-5 Floor and sample-7 wall) 13 FE-SEM micrograph & EDS spectra of (a) smast-7 wall and (b) 75 smast-5 floor 14 Nutrient agar plate showing the zones of inhibition against the 76 clinical isolates 15 Phylogenetic tree of all four species with related sequences in NCBI 77-78 16 Effect of time of incubation, pH and temperature on the growth and 79 antimicrobial activity of B. licheniformis KC2-MRL against M. luteus, S. aureus, Klebsiella and E. coli 17 Zone of inhibition of our four antibiotic producing strains (Serratia 80 sp. KC1-MRL, Bacillus licheniformis KC2-MRL, Bacillus sp. KC3- MRL and Stenotrophomonas sp. KC4-MRL) against selected antibiotics in mm. 18 Comparison of FTIR spectra of control (Bacitracin) and the 81 antibacterial compound produced by B. licheniformis KC2-MRL 19 Fourier-transform infrared spectra from control and different media 112 after incubation at 37°C for one month 20 Scanning electron microscopy of low-density polyethylene samples 114 under specified treatment after incubation with bacterial consortia at 37°C for one month. 21 Limestone cave (Kashmir Smast Pirsai Mardan, Pakistan) 126 22 Evolutionary relationships of taxa Lysinibacillus sphaericus KC5- 130 MRL. 23 Effect of size of inoculum on the growth of Lysinibacillus 131 sphaericus KC5-MRL and production of lipase. 24 Lipolytic, amylolytic and proteolytic activity of Lysinibacillus 132 sphaericus KC5-MRL at 30°C 25 Stability of crude extracts of lipase, amylase and protease of 134 Lysinibacillus sphaericus KC5-MRL at different pH 26 Stability of crude extracts of lipase, amylase and protease of 136 Lysinibacillus sphaericus KC5-MRL at different temperature 27 Effects of detergents on the lipolytic, amylolytic and proteolytic 137 activity of isolate Lysinibacillus sphaericus KC5-MRL 28 Effects of metal ions on lipase, amylase and protease activity of 138 isolate Lysinibacillus sphaericus KC5-MRL 29 Effects of organic solvents on lipase, amylase and protease activity 139 of Lysinibacillus sphaericus KC5-MRL 30 Effect of inhibitors on lipase, amylase and protease activity of 139 Lysinibacillus sphaericus KC5-MRL 31 Molecular Phylogenetic analysis by Maximum Likelihood method 154 32 Calcium precipitates induced by bacteria in crystals form A) 155 Paracoccus Limosus, B) Brevundimonas naejangsanensis. 33 Compound microscopy of precipitates produced at 25°C. 155 34 Compound microscopy of precipitates produced at pH 5. 156 35 Compound microscopy of precipitates produced after 20 days of 156 incubation. 36 Electron microscopy at different wavelength (A) at 500 μm, (B) at 157 200 μm, (C) at 100 μm, (D) 50 μm, (E) 1 micro meter. 37 FTIR analysis of CaCO3 with control. 158 38 XRD analysis of the polymorphs of Calcium carbonate. 165 39 Mn cycle of oxidation states found in nature 175 40 Four possible mechanisms of Mn+2 oxidation by bacteria. 176 41 Enzymatic pathway of Mn(II) oxidation 177 42 Kashmir Smast (Cave) entrance zone 180 43 Speleothems isolated from Kashmir smast (Cave) 181 44 Initial screening of Mn(II) oxidizing bacterial strains from cave soil. 187 45 Stereoscopy of the isolates. 188 46 DNA bands of Mn(II) oxidizing isolates 188 47 Phylogenetic anaylsis by Maximum Likelihood method 189 48 Growth curves of Bacillus pumilus C3 at 30oC and 25oC (No 190 o o Mn600, 30 C and No Mn600, 25 C), in the presence of MnCl2 (Mn600, 30oC), after adding 1 and M of Super Oxides Dismutase (SOD) (Mn+SOD1,600. Mn+SOD5,600), and in the presence of 100M Calcium acetate (Mn+Ca, 600) 49 Growth curves of Bacillus Safensis C6 at 30oC and 25oC (No 191 o o Mn600, 30 C and No Mn600, 25 C), in the presence of MnCl2 (Mn600, 30oC), after adding 1 and M of Super Oxides Dismutase (SOD) (Mn+SOD1,600. Mn+SOD5,600), and in the presence of 100M Calcium acetate (Mn+Ca, 600). 50 Growth curves of Bacillus pumilus C7 at 30oC and 25oC (No 192 o o Mn600, 30 C and No Mn600, 25 C), in the presence of MnCl2 (Mn600, 30oC), after adding 1 and M of Super Oxides Dismutase (SOD) (Mn+SOD1,600. Mn+SOD5,600), and in the presence of 100M Calcium acetate (Mn+Ca, 600). 51 Growth curves of Bacillus cereus C8 at 30oC and 25oC (No Mn600, 193 o o 30 C and No Mn600, 25 C), in the presence of MnCl2 (Mn600, 30oC), after adding 1 and M of Super Oxides Dismutase (SOD) (Mn+SOD1,600. Mn+SOD5,600), and in the presence of 100M Calcium acetate (Mn+Ca, 600). 52 Growth curves of Bacillus acidiceler C11 at 30oC and 25oC (No 194 o o Mn600, 30 C and No Mn600, 25 C), in the presence of MnCl2 (Mn600, 30oC), after adding 1 and M of Super Oxides Dismutase (SOD) (Mn+SOD1,600. Mn+SOD5,600), and in the presence of 100M Calcium acetate (Mn+Ca, 600). 53 Variation in Mn(II) oxidation at different pH and Ca+2 ion 195 concentration. 54 196 Mn(II) oxidation capacity and Mn(III, IV) oxide concentration as a 197 function of reaction time in C rich media K-medium. The ages of the 198 Mn oxide are from 4 h to 36 h. 199 55 Effect of metals on Mn oxide production by cavernicoles after 24 h 200 of incubation. Acknowledgements Praise to ALMIGHTY ALLAH, whose blessings enabled me to achieve my goals. Tremendous praise for the Holy Prophet Hazrat Muhammad (Peace Be upon Him), who is forever a torch of guidance for the knowledge seekers and humanity as a whole. I have great reverence and admiration for my research supervisor, Dr. Fariha Hasan, Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan, for her scholastic guidance, continuous encouragement, sincere criticism and moral support throughout the study. Her guidance helped me in all the time of research and writing of this thesis, with her patience and immense knowledge. I do not find enough words to express my heartfelt gratitude for Dr.
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