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Microbiological Analysis of Two Glacier Samples from North Sikkim Sikkim

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Microbiological Analysis of Two Glacier Samples from North Sikkim Sikkim Microbiological Analysis of two Glacier samples from North Sikkim A Thesis Submitted To Sikkim University (A Central University established by an Act of Parliament of India, 2007) In Partial Fulfilment of the Requirement for the Degree of Doctor of Philosophy By Mingma Thundu Sherpa Department of Microbiology School of Life Sciences December, 2018 1 AKNOWLEDGMENT I am very grateful to acknowledge and would like to thank my supervisor, Dr. Nagendra Thakur, Assistant Professor, Department of Microbiology, Sikkim University and Pos doctorate fallow KIST (Korean Institute of Science and Technology), South Korea for all his ideas, suggestions, inputs and encouragement which he had provided during my work. He had instilled a sheer faith and belief in me to accomplish my work. I would like to acknowledge and thank our Dean, school of life Science Prof. (Dr.) Jyoti Prakash Tamang (Professor, Department of Microbiology), for being such a kind and helpful person, who has been a great facilitator for our Departmental progress. I would like to thank our Head of the Department (H.O.D), Dr. Hare Krishna Tiwari, Associate Professor, for all his encouragement and support, which he had provided during my work. I also thankful to all my dear teacher - Dr. Buddhiman Tamang, Dr. Bimala Singh, Dr. Anil Kumar Verma - Assistant Professors, from the Department of Microbiology, Sikkim Universities, for their inspiration, help and suggestions throughout my Ph.D work. I would like to thank dear Mrs. Radha didi (Radha Basnett, Technical Assistant), Mr. Pukar Biswakarma (Laboratory Attendant)and Mr. Gagan Sen Chettri (Former Lab. Incharge) Department of Microbiology, Sikkim University for providing me with all the essential instruments, Media, Chemicals, Reagents and Glass apparatus in good condition, to work with. I would like to thank Dr. Uttam Lal (Assistant Professor, Department of Geography and Natural Resources, Sikkim University); Dr. Rakesh Kumar Ranjan (Assistant Professor, Department of Geology, Sikkim University) and Dr. Smiriti Basnet (Project Coordinator, Indian Institute of Science, IIS, Bangalore) for all their kind help and contributions for my thesis work. I would like to thank Department of Science & Technology, Govt. of India for providing me with the JRF position and funding my work. MINGMA THUNDU SHERPA i This work is not possible without my Senior Mr. Ishfaq Nabi Najar and my friend Mr. Sayak Das, both were continuously helping me throughout the work. This work would have not been possible without the research permission and support from Department of Forest, Government of Sikkim and Sikkim Police, for their unflinching support to carry out the field survey. Special thanks are due my colleagues - Pooja Pradhan, Meera Ongmu Bhutia, Nilu Pradhan and Anu Anupama. My thanks are extended to Ashish Kumar Singh, Lalit Kumar Chaurasia and Dr. Saurav Das for numerous tea session companies, for their encouragement, memorable times spend together and support. I am very grateful to Sonam Lachungpa and his family, Lachung North Sikkim for their valuable support during my sample collection and lodging. Without parents blessing, their believe, and love, it would have been impossible for me to come and do my research. I want to thank my mother Nim Lhamu Sherpa (ama), father Rinzi Sherpa (appa), brothers Phurba Dorjee Sherpa, Passang Dorjee Sherpa, Pem Tshering Sherpa, Mingma Norbu Sherpa and my sisters Phur Diki Sherpa, Nim Diki Sherpa and my Bhanji Aisha Chettri for their love and constant encouragement. I would like to thank the Department of Microbiology, School of Life Sciences, Sikkim University, for providing excellent working conditions and providing me the laboratory spaces for my research. Last but not the least, our team which was always with me during my best and worst of times. Research cannot be done individually, and hence without the help and unconditional love, moral support, sharing work and help throughout all my field work, sample collection and setting up experiments, our team was there for me and even after completion of my work, they will be with me with their help and love. I would like to express my heartfelt acknowledgement to Sayak, Ishfaq and Nagendra Sir – MY TEAM forever!!!. ii iii iv v CONTENTS Page No. Acknowledgement i-ii Contents iii-ix List of Tables x-xii List of Figures xiii-xiv Abbreviation 1. INTRODUCTION 1-11 2. RATIONALE AND SCOPE 12-16 2.1. Aims and Objectives 16-17 3. REVIEW OF LITERATURE 18-41 3.1. General description of glacier 18 3.2. Formation of mountain glacier 20 3.3. Microbial life in the glacier 21 3.4. Microorganisms immured in glacier ice 23 3.5. Methodologies to study bacterial diversity 25 3.6. Bacterial diversity in glacial ice 28 3.7. Adaption of psychrophiles to low temperature 30 3.8. The biotechnological significance of Psychrophiles 32 3.9. Application of Psychrophiles 34 3.10. Bioremediation 34 vi 3.11. Detergent industry 35 3.12. Food Industry 36 3.13. Textile Industry 37 3.14. Cryozymes/Psychrophilic enzymes 38 3.15. Cold-adapted lipase 38 3.16. Cold-adapted protease 39 3.17. Cold-active amylases 40 4.0. MATERIALS 42-48 4.1. Materials used for measuring and mapping of the 42 geographical features of the sampling sites 4.2. Materials used for sample collection 42 4.3. Instrument used for the determination 42 of a physical parameter of the glacial samples 4.4. Instrument used for the determination of the 42 Chemical Analysis of the glacial samples 4.5. Materials used for the Isolation and Cultivations of 42 the microorganisms 4.5.1. Microbiological Media 42 4.5.2. Chemicals 43 4.5.3. Materials for the staining of the microorganisms 44 4.5.4. Materials for the biochemical Analysis of 44 the microorganisms 4.5.5. Antibiotic used for Antibiotic Assay 45 4.6. Laboratory Equipment and Instruments 45 4.7. Glassware 46 4.8. Materials used for the Molecular Characterization 47 4.9. Others Materials 47 4.10. Software used for data analyses 48 vii 5.0. METHODS 49-69 5.1. Mapping of the sampling sites 49 5.2. Glacier ice sampling 49 5.3. Physicochemical Analysis of the glacial samples 50 5.3.1. Methods used for the analysis and determination 50 of the physical characteristics of the glacial samples 5.3.2. Methods for the determination of the chemical 50 characteristics of the glacial samples 5.3.3. Statistical analysis of chemicals 51 5.4. Microbial Analysis of the glacial samples 51 5.4.1. Enumeration of the microbial load by total plate count 51 5.4.2. Isolation and culturing of bacteria 52 5.4.3. Characterization of the bacterial isolates from 52 glacier ice samples 5.4.3.1. Cultural characteristics and morphology 52 5.4.4. Growth profile at various physical parameters 53 5.4.4.1. Tolerance to different degrees of Temperature 53 5.4.5. Tolerance to different degrees of pH 54 5.4.6. Tolerance to different concentrations of NaCl 54 5.5. Biochemical characterization of the bacterial isolates 55 5.5.1. Carbohydrate Fermentation Test 55 5.5.2. Catalase Test 55 5.5.3. Protease Test 55 5.5.4. Starch Hydrolysis Test 55 5.5.5. Lipase Test 55 5.5.6. Gelatin Hydrolysis Test 55 5.6. Determination of the proteolytic and amylase activity 56 viii 5.6.1. Screening of protease and amylase producing 56 bacterial strains 5.6.2. Enzyme production 56 5.6.3. Estimation of enzymatic activity 57 5.7. Identification of bacterial isolates by Culture-dependent 57 Techniques 5.7.1. Genomic DNA extraction 57 5.7.2. Polymerase Chain Reaction (PCR) Amplification 59 5.7.3. Purification of PCR products 60 5.7.4. DNA sequencing 60 5.7.5. Statistical analysis 61 5.8. Fatty acid methyl-ester Analysis 62 5.9. Identification of Putative Novel Psychrotolerant bacteria 63 5.9.1. Morphological classification 63 5.9.2. SEM (Scanning Electron Microscope) 63 5.9.3. Growth Profile at various physical parameters 63 5.9.4. Biochemical characterization (by manual methods) 64 5.9.5. Biochemical characterization (BIOLOG) 64 5.9.6. Fatty Acid Methyl Ester (FAME) analysis 64 5.10. Identification of bacterial diversity by 64 Culture-independent techniques 5.10.1. Phospholipids fatty acid (PLFA) analysis 64 5.10.2. Next Generation Sequencing 64 5.10.2.1. Metagenomic DNA extraction 64 5.10.2.2. Metagenomic Library preparation and Sequencing 65 5.10.2.3. Data availability and accession number 65 5.10.2.4. Bioinformatics analysis 65 5.10.2.5. Functional analysis of the Metagenome data 66 5.10.2.6. Antibiotic Resistance Genes (ARGs) analysis 66 5.10.2.7. Metal Resistance Genes (MRGs) analysis 67 ix 5.11. Antibiotic Sensitivity of Isolates 67 5.11.1. The minimum inhibitory concentration 68 of antibiotics (MICs) 5.12. Heavy metal tolerance to isolates 68 5.12.1. Screening and assessment of metal toxicity 68 5.12.2. Tube dilution method 69 6.0 RESULTS 70-143 6.1. Description of sampling sites 70 6.2. Sampling and physiochemical analysis of Samples 71 6.2.1. Sample collection and Pre-treatment 71 6.2.2. Physical analysis of samples 72 6.2.3. Chemical Analysis of the glaciers samples 73 6.2.4. Statistical and comparative analysis of glacier samples 76 6.3. Culture-dependent Analysis from glaciers samples 77 6.3.1. Isolation of bacterial strains from glaciers 77 6.3.2. General Morphology of the bacterial isolates 77 6.3.3. Staining 80 6.3.4. Growth profiles at different physical parameters 83 6.4. Biochemical characterization of the isolates 86 6.4.1. Carbohydrate Fermentation assessment 86 6.4.2.
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