Culturable Diversity of Psychrophilic Bacteria from Different Glaciers of Karakoram Mountain Range and Role of Cell Membrane Fatty Acids in Cold Adaptation By Noor Hassan Department of Microbiology Faculty of Biological Sciences Quaid-I-Azam University Islamabad, Pakistan 2020 Culturable Diversity of Psychrophilic Bacteria from Different Glaciers of Karakoram Mountain Range and Role of Cell Membrane Fatty Acids in Cold Adaptation A thesis Submitted in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY IN MICROBIOLOGY By Noor Hassan Department of Microbiology Faculty of Biological Sciences Quaid-I-Azam University Islamabad, Pakistan 2020 In the name of ALLAH, Most Gracious, Most Merciful. DEDICATION Dedicated to My Beloved Parents And Wife Author’s Declaration I Mr. Noor Hassan hereby state that my Ph.D thesis titled “Culturable Diversity of Psychrophilic Bacteria from Different Glaciers of Karakoram Mountain Range and Role of Cell Membrane Fatty Acids in Cold Adaptation” is my own work and has not been submitted previously by me for taking any degree from Quaid-i-Azam University, Islamabad, Pakistan. At any time if my statement is found to be incorrect even after I Graduate, the University has the right to withdraw my Ph.D degree. Mr. Noor Hassan Date: 22-07-2020 Plagiarism Undertaking “Culturable Diversity of Psychrophilic Bacteria from Different Glaciers of Karakoram Mountain Range and Role of Cell Membrane Fatty Acids in Cold Adaptation” is solely my research work with no significant contribution from any other person. Small contribution / help wherever taken has been duly acknowledged and that complete thesis has been written by me. I understand the zero tolerance policy of the HEC and Quaid-i-Azam University towards plagiarism. Therefore I as an Author of the above titled thesis declare that no portion of my thesis has been plagiarized and any material used as reference is properly referred/cited. I undertake that if I am found guilty of any formal plagiarism in the above titled thesis even after award of Ph.D degree and that HEC and the University has the right to publish my name on the HEC/University Website on which names of students are placed who submitted plagiarized thesis. Signature: Name: Mr. Noor Hassan Certificate of Approval This is to certify that the research work presented in this thesis, entitled titled “Culturable Diversity of Psychrophilic Bacteria from Different Glaciers of Karakoram Mountain Range and Role of Cell Membrane Fatty Acids in Cold Adaptation” was conducted by Mr. Noor Hassan under the supervision of Prof. Dr. Fariha Hasan. No part of this thesis has been submitted anywhere else for any other degree. This thesis is submitted to the Department of Microbiology, Quaid-i-Azam University, Islamabad in partial fulfillment of the requirements for the degree of Doctor of Philosophy in field of Microbiology. Student Name: Mr. Noor Hassan Signature: Examination Committee: a) External Examiner 1: Signature: Dr. Ijaz Ali Associate Professor Department of Microbiology COMSATS University Park Road Islamabad b) External Examiner 2: Signature: Dr. Qaiser Mahmood Associate Professor Department of Environmental Sciences COMSATS University Abbottabad Campus Abbottabad Supervisor Name: Prof. Dr. Fariha Hasan Signature: Name of HOD: Dr. Aamer Ali Shah Signature: List of Contents Sr. # Titles Page # 1. List of Tables……………………………………….. i 2. List of Figures……………………………………... iii 3. List of Abbreviations….……………………..…..... v 4. Acknowledgements……………………………….. vii 5. Summary...………………………………………… ix 6. Chapter 1…………….…………………………..... 01 7. Chapter 2...………………………………….…...... 18 8. Chapter 3…………………………………..……… 58 9. Chapter 4……………………………………..….. 104 10. Chapter 5………………………………………… 135 11. Chapter 6………………………………….…..…. 160 12. Overall Conclusions……...………………..…..… 192 13. Future Prospects……………………………...…. 194 14. Appendices………………………………….......... 195 List of Tables Page No Titles No. 1.1 An overview of dissimilarities between psychrophiles and psychrotrophs 2 Distribution of major bacterial phyla in glaciers situated in Polar and non- 1.2 4 polar regions 3.1 Colony forming unit (CFU)/mL or g and coordinates of samples 67 The primers used for the PCR amplification of 16S rRNA and 3.2 68 rpoB genes of bacterial isolates 3.3a Major ion content of glacial samples 71 Total organic carbon, total nitrogen and total phosphorus content of glacial 3.3b 72 samples The studied bacterial species with corresponding homologous strains and 3.4a 73 their accession numbers isolated from Ghulmet glacier The studied bacterial species with corresponding homologous strains and 3.4b 75 their accession numbers isolated from Ghulkin glacier The studied bacterial species with corresponding homologous strains and 3.4c 76 their accession numbers isolated from Hopar glacier An overview of quantified and identified pigments produced by bacterial 3.5 83 isolates List of bacterial strains producing straight chain monounsaturated fatty 4.1a 110 acids as major group of cell membrane fatty acids List of bacterial strains producing branched chain fatty acids as major 4.1b 111 group of cell membrane fatty acids Correlations between various temperature and single type of cell 4.2a 114 membrane fatty acid (Gram-negative bacteria) Correlations between various temperature and single type of cell 4.2b 116 membrane fatty acid (Gram-positive bacteria) i | P a g e Growth characteristics of the Serratia marcescens HI6 on different 5.1 142 physiological parameters Distribution and percentage of fatty acids in the cell membrane of the 5.2 146 Serratia marcescens HI6 The quantitative overview (with triplicates data and standard deviations) 5.3 147 of cell membrane fatty acids of Serratia marcescens HI6 Details of the primers used for PCR amplification of 16S-23S intergenetic 6.1 167 segment of the isolate CHS1 The resemblance directory of the isolate CHS1 with respective 6.2 169 homologous species Growth characteristics of the cyanobacterium isolate CHS1 on different 6.3 171 physiological parameters 6.4 Different pigments produced by Nodularia spumigena CHS1 172 6.5 The profile of cell membrane fatty acids of Nodularia spumigena CHS1 175 The quantitative overview (with triplicates data and standard deviations) 6.6 176 of cell membrane fatty acids of cyanobacterium CHS1 ii | P a g e List of Figures Page No Titles No. Geographic location of Indus River connected with Gilgit and Hunza 1.1 8 Rivers in Karakoram Mountain Range, Pakistan An overview of the HKKH, largest reservoirs of glaciers outside Polar 2.1 19 regions 2.2 A typical graphic representation of Valley glacier with different parts 20 2.3 An overview of adaptation mechanisms in psychrophilic bacteria 30 2.4 Structures of important fatty acids produced by psychrophilic bacteria 32 2.5 Biosynthetic pathway of fatty acids proposed for E. coli 34 Structures of carotenoid pigments, biosynthetic pathway and genes 2.6 37 involved in the synthesis of carotenoids Topographic representation of sampling sites located in Karakoram 3.1 62 Mountain Range, Pakistan 3.2 HPLC chromatograph of known standards of pigment 78 The phylogenetic relationship of bacteria isolated from Ghulmet 3.3a glacier with their homologous bacterial species constructed by the 79 Maximum Likelihood method The phylogenetic relationship of bacteria isolated from Ghulkin 3.3b glacier with their homologous bacterial species constructed by the 80 Maximum Likelihood method The phylogenetic relationship of bacteria isolated from Hopar glacier 3.3c with their homologous bacterial species constructed by the Maximum 81 Likelihood method iii | P a g e Pearson correlation of violacein and bacterioruberin produced by 3.4 85 bacteria species at different temperature Gas chromatography/mass spectroscopy chromatograph/profile of 4.1 113 external FAMEs standards with retention times Temperature dependent accumulation of principle fatty acids groups 4.2a 117 of gram-negative bacteria Temperature dependent accumulation of principle fatty acids groups 4.2b 121 of gram-positive bacteria Distribution of major types of fatty acids in cell membrane of Gram 4.3a 123 negative bacteria at different temperatures Distribution of major types of fatty acids in cell membrane of Gram 4.3b 124 positive bacteria at different temperatures The colony morphology of the isolate HI6 with production of red 5.1 141 pigment Analysis of evolutionary relationship and relatedness of Serratia 5.2 143 marcescens HI6 by Maximum Likelihood method Fourier Transform infrared (FTIR) spectrum of FAME extracted from 5.3 144 the Serratia marcescens HI6 Distribution of individual fatty acids in the cell membrane of Serratia 5.4 145 marcescens HI6 Microscopic observation of isolate CHS1 with light microscope 6.1 170 Olympus CX 40 Phylogenetic analysis of the Nodularia spumigena CHS1 based on 6.2 172 16S-23S intergenetic region by Maximum Likelihood method Major fatty acids components of cell membrane of the Nodularia 6.3 174 spumigena CHS1 iv | P a g e List of Abbreviations AA Arachidonic acid ACP Acyl carrier protein ɑ-LA ɑ-Linolenic acid ATP Adenosine tri phosphate b-FAs Branched chain fatty acids CFB Cytophaga-Flavobacteria-Bacteroides CFU Colony Forming Unit CHS Cyanobacterium Hopar Sediment DCGs Debris-covered glaciers DHA Docosahexaenoic acid DMADP Dimethylallyl diphosphate EPA Eicosapentaenoic acid EPS Extracellular polymeric substances ER East Rongbuk FAMEs Fatty acid methyl esters FAS Fatty acid synthases FTIR Fourier Transform infrared GC/MS Gas chromatography/mass spectroscopy