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Bacterial Flora of Manasbal Lake, a Freshwater Ecosystem of Kashmir Valley

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Bacterial Flora of Manasbal Lake, a Freshwater Ecosystem of Kashmir Valley Bacterial Flora of Manasbal Lake, a Freshwater Ecosystem of Kashmir Valley Dissertation Submitted in Partial Fulfillment of the Requirements for the Award of Degree of MASTER OF PHILOSOPHY (M.PHIL.) IN ENVIRONMENTAL SCIENCE By Sana Shafi M.Sc. B.Ed. Under the Joint Supervision of Dr. Manzoor A. Shah Prof. Azra N. Kamili Sr. Assistant Professor Head/ Director Department of Botany Environmental Science/CORD (Co-supervisor) (Supervisor) POST GRADUATE DEPARTMENT OF ENVIRONMENTAL SCIENCE Faculty of Physical and Material Sciences UNIVERSITY OF KASHMIR SRINAGAR-190006, KASHMIR August, 2012 Phone: Office: 0194-2420078, 2420405, Ext. 2155 email: [email protected]; [email protected] Department of Environmental Science University of Kashmir, Srinagar -190006 No. Dated: Certificate This is to certify that the M. Phil. Thesis entitled “Bacterial flora of Manasbal Lake, a fresh water ecosystem of Kashmir Valley” is the original research work carried out by Ms Sana Shafi, as whole time M. Phil. research scholar in Department of Environmental Science, University of Kashmir, Srinagar. This work has been carried out under our joint supervision and has not been submitted to this University or to any other University so far and is submitted for the first time to the University of Kashmir. It is further certified that this thesis is fit for submission for the degree of Master of Philosophy in Environmental Science and the candidate has fulfilled all the statutory requirements for the completion of the M. Phil. Programme. Dr. Manzoor A. Shah Prof. Azra N. Kamili Sr. Assistant Professor Head/Director Department of Botany Department of Environmental University of Kashmir Science/ CORD Co-Supervisor University of Kashmir Supervisor Head/Director Environmental Science/CORD, University of Kashmir Srinagar-190006. i Dedicated to My Parents CONTENTS Chapter No DESCRIPTION PAGE NO. Certificate i Acknowledgement ii-iii ABSTRACT iv-vi 1. INTRODUCTION 1-9 1.1. Objectives 7 1.2. Study Area 8-9 2. REVIEW OF LITERATURE 10-39 2.1. Bacterial diversity 11-17 2.2. Ecological factors influencing bacterial 18-25 diversity 2.3. Bacteria as indicator of pollution 26-32 2.4. Role of bacteria in bioremediation of aquatic systems 32-39 3. MATERIAL AND METHODS 40-47 3.1. Cleaning of glassware 40 3.2. Sterilization 41 3.3. Preparation of Media and Diluent 42-43 3.4. Dispensing Of Media 43 3.5. Culture Techniques 43 3.6. Serial Dilution Technique 43 3.7. Inoculation 44 3.8. Incubation 44 3.9. Enumeration of Colonies 44 3.10. Pure Culture Techniques 45 3.11. Identification 45-47 3.12. Motility of bacteria 47 4. RESULTS 48-82 4.1. Distribution of colonies 48-51 4.2. Total coliform 51-52 4.3. Biochemical tests 52-55 4.4. Temperature and pH 55 5. DISCUSSION 83-93 5.1. Distribution of colonies 83-84 5.2. Total coliform 85-86 5.3. Biochemical tests 86-93 5.4. Temperature and pH 93 6. CONCLUSIONS 94-96 7. BIBLIOGRAPHY 97-128 LIST OF TABLES Table Page Description No. No. Table 1. Colony Morphology of Different Bacterial Isolates 56-57 Percentage of Gram Positive and Gram Negative Table 2. 58-59 Bacterial Strains Table 3. Shape Wise Percentage of Different Bacterial Strains 60-61 Percentage Occurrence of Gram Positive and Gram Table 4. 62 Negative Bacteria Table 5. Monthly Distribution of Bacterial Colonies (cfu/ml) 63 Table 6. Total Viable Count of Bacteria at Site I 64 Table 7. Total Viable Count of Bacteria at Site II 64 Table 8. Total Viable Count of Bacteria at Site III 64 Table 9. Total Viable Count of Bacteria at Site IV. 65 Total Coliform Count of Different Water Samples Table 10. 65 (MPN/100ml) Category Wise Distribution of Coliform Count Table 11. 66 (MPN/100ml) Biochemical Results of Bacterial Species for Table 12. 67 Enterobacteriaceae Family Biochemical Results of Bacterial Species for Gram Table 13. 68 Negative Rods Table 14. Total Viable Count of the Identified Species at Site I 69-70 Table 15. Total Viable Count of the Identified Species at Site II 71-72 Table 16. Total Viable Count of the Identified Species at Site III 73-74 Table 17. Total Viable Count of the Identified Species at Site IV 75-76 Table 18. Comparative Bacterial Analysis at Different Sites 77 Total Colony Count and Percentage Occurrence of Table 19. 78 Bacterial Species Monthly Colony Count of Bacterial Species at Different Table 20. 79 Sites Table 21. Monthly Shannon Index (H) at Different Sites 79 Table 22. Water Temperature (°C) Recorded at Four Sites 81 Table 23. pH of Water Recorded at Four Sites 82 LIST OF FIGURES Figure No. Description Page No. Percentage Occurrence of Gram Positive and Fig. 1 62 Gram Negative Bacteria Monthly Distribution of Bacterial Colonies Fig. 2 63 (cfu/ml) Total Coliform Count of Different Water Fig. 3 66 Samples (MPN/100ml) Shannon Index (H) of Different sites in Different Fig. 4 80 Months Site Wise Shannon index (H) of Bacterial Fig. 5 80 Communities Fig. 6 Water Temperature Recorded at Four Sites 81 Fig. 7 pH of Water Recorded at Four Sites 82 LIST OF PHOTOGRAPHS Photograph Description No. 1. Map showing different study sites of Manasbal Lake Photographs showing different study sites of Manasbal 2. lake Photographs showing different isolation techniques 3. Photographs showing different Grams nature and shape 4. of bacteria Photographs showing Biochemical test kit for 5. Enterobacteriaceae Photographs showing Biochemical test kit for Gram 6. Negative Rods List of Abbreviations APHA : American Public Health Association BCC : Bacterial Community Composition CFU : Colony Forming Unit DOC : Dissolved Organic Carbon EMB Agar : Eosine Methylene Blue Agar Fig : Figure m.a.s.l : Meters above sea level lb/inch2 : Pound per inch square g : Gram Hrs : Hours mg : Milligrams °C : Degree Centigrade ml : Millilitre MPN : Most Probable Number NaCl : Sodium Chloride PCBs : Poly-chloro-biphenyls PCR : Polymerase Chain Reaction spp. : Species UNESCO : United Nations Educational Scientific and Cultural organisation UNICEF : United Nations International Children’s Emergency Fund USEPA : United State Environmental Protection Agency -ve : Negative +ve : Positive WHO : World Health Organisation ACKNOWLEDGEMENTS All praises to Almighty Allah (most merciful and beneficent) who by His grace helped me to successfully complete M.Phil programme with great zeal and zest. I take this opportunity to express my deep sense of gratitude and indebtedness towards my teacher and guide Prof. Azra N. Kamili, Head, Department of Environmental Science / Director CORD, University of Kashmir, for suggesting me the work of vital interest. This study was conducted at her initiative and under her supervision. I am thankful to her for her guidance, invaluable suggestions, enthusiastic encouragement and necessary research facilities provided during the course of my studies. I must confess that without her constant endeavour in guiding me in completion of this work, it could not have been completed successfully. I wish to express my deep sense of gratitude and indebtedness to Dr. Manzoor A. Shah, Sr. Assistant Professor Department of Botany, University of Kashmir, for his enthusiastic guidance, kind supervision, continued encouragement, generous assistance, timely cooperation, valuable suggestions and constructive criticism during the course of present study. I also express my profound gratitude to Prof. A. R. Yousuf (former Dean Academic Affairs), Prof. A. K. Pandit (CORD/Environmental Science) and Prof. G. A. Bhat (CORD/Environmental Science) for their moral support, useful suggestions and continuous encouragement all through the course of study. ii My sincere and deep regards are due to Dr. Ruqaya Nazir, Assistant Professor (CORD) Microbiology Laboratory Incharge for her constant encouragement and kind behaviour. Thanks are also due to other technical and non technical staff members of CORD, especially Mrs Bilquis Qadri (Scientific Officer), Ms Rubiya Dar (Sr. Technical Assistant), Ms Sumaira Tyub (Sr. Technical Assistant) and Mr Javaid A. Javaid (Jr. Technical Assistant) for their cooperation, timely help and whole hearted support. The successful completion of the research programme was possible only because of the active cooperation of my friends and colleagues. I would like to put on record my thanks to Scholars of CORD, in general, and Microbiology Laboratory, in particular, namely Mr Suhaib A. Bandh, Mrs Samira Amjid, Ms Humera Nissa, Ms Nowsheen Shameem Mr Javaid A. Parray, Mr Bashir A. Lone and Mr Gowhar H. Dar who rendered full cooperation and support during the research programme without which conduct of field study and data analysis would have been rather difficult. I am also thankful to my friends Ms Rahila Bashir, Ms Tabasum Yaseen, Ms Aalia Ismat, Ms Sana Mehraj and Mrs Sakeena Waseem for their help and fruitful guidance. I fail in my duty if I don’t make a special mention here of my parents and my sister, Ms Shabnum and other family members who encouraged me all throughout the research work. Sana Shafi iii Abstract Abstract icro-organisms have been used for a long time as indicators of water quality and total coliform bacteria have been commonly M used to assess potential contamination of drinking and swimming water with pathogenic bacteria of intestinal origin coming from point source discharges, such as raw sewage, storm water, combined sewer overflows, effluents from wastewater treatment plants, industrial sources and non-point source discharges, such as agriculture, forestry, wildlife, and urban run-off. The obtained data in this study reflect the importance of microbiological monitoring and reinforce the need to implement environment protection programs, especially related to pathogenic species. The majority of bacteria isolated were recognized as human pathogens or potential human pathogens. The data was obtained by the bacteriological analysis of water sample taken from Manasbal Lake on monthly basis from four different microhabitats by plating the different dilutions on a solidified culture medium in petri dishes.
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