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Studies on Microbial Biodiversity of Acidophilic Heterotrophs in Acid Rock Drainage Samples of Eastern Himalaya

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Studies on Microbial Biodiversity of Acidophilic Heterotrophs in Acid Rock Drainage Samples of Eastern Himalaya Studies on microbial biodiversity of acidophilic heterotrophs in Acid Rock Drainage samples of Eastern Himalaya THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN SCIENCE (BIOTECHNOLOGY) OF THE UNIVERSITY OF NORTH BENGAL Suparna CBiiowa( Microbial Biotechnology Laboratory Department of Biotechnology University of North Bengal 2011 'lTie onfy way offindi11f1 tlie Bmits oftlie possi6(e is 6y going 6eyontf tfiem into tlie impossi6fe. -l/.rtfiur C Cfar&e Ae/CNOWI.B'DQBMBN7S This is a solemn opportunity to express my veneration to the entire magnanimous fraternity to those who directly or indirectly have contributed or associated to this work and made it possible. No words or mere accolades are big enough to express my gratitude towards, the person who stood rock solid beside me during the entire tenure of my research career, yes he is my guide, my supervisor, my guardian Dr. Ranadhir Chakraborty. His guidance, continuous inspiration and boundless optimism encouraged me to pull the worl< and give it a meaningful conclusion. Not even a part of the work would have reached its culmination without him. I owe special thanks and gratitude to my teachers Mr. T. Gopalan and Mr. Balwinder Singh Bajwa, who seeded and cultured the scientific research aptitude within me. · Right from my first day in the Department o(Biot~chnology, I had the opportunity to gain some good advice and wisdom from my teachers Dr. Shilpi Ghosh and Dr. Dipanwita Saha. I express my sincere thanks to my teachers Prof. B.N. Chakraborty, Prof. P.K. Sarkar, Prof. A.P. Das, Prof. Usha Chakraborty, Prof. P. K. Basu, Dr. Aniruddha Saha, Dr. Arnab Sen, Sri Palash Mandai and all other teachers and non-teaching staffs of the Department of Botany, N.B.U. for their valuable advice and genuine assistance. I got overwhelming support from the Department of Botany, Zoology, Computer Science and Chemistry of University of North Bengal, in all possible forms starting from free flow of logistic amenities to sharing of infrastructures, technical know-how and academic knowledge. So, I record my special thanks to Soubasya Sur, Manpreet Bajwa, Monojeet da, Bikash da, Sikta di, Sir C. R. Nayak, and Dr. A. K. Nanda. I am also grateful for financial assistance in the form of fellowship provided by the DBT research grant (BT/PR-7164/BCE 08/448/2006), Govt. of India. When I started off as a Ieamer during my research period , trying to imbibe every moments as I slowly progressed, the journey resurfaced as I started learning every step of my accomplished job from my senior lab-mates Shriparna di, Bhaskar da, and Anirudra da. Others followed suit with equal fervour, romping me home with all their diligent enthusiasm to the best of their abilities Arvind Kumar, Bipransh Tiwary, Amit Mandai, Dhananjay Kumar, Prashantee Singh, Debjani Sarkar, Rituparna Sarkar, and Sonam Bhutia fills up this void to create an all pervading atmosphere of compassionate vigour. I would also like to thank Papai Singha, Subhash and Bishu who also helped me sail smoothly by providing continues support and assistance. Special thanks to Kamal, Manab, Madhumita, Moushree, Gargi, Anasooya, and other research scholars for their help and for providing healthy ambience during my work. Few people have a very special place in our lives, sharing a bond which is none others can prove to be stronger off. Although not directly involved in any scientific contributions my buddies Sanghita, Amrita, Soumen, Swagata, Abhijit, Kallol, Biswajit, Sudipta, Deep, the list goes on .. , have always provided me the light atmosphere which is always a boon in a profession like ours. Spoilt to the brink with an overdose of unadulterated fun and enjoyment, I still treasure the quality time spent with them who had backed me with conviction. It will perhaps be improper to name individuals since the list of philanthropists who had bailed me out at times of desperate need is so long that it involves the risk of special persons get omitted. I should convey my thanks to all my students. Nevertheless, I must offer my special accolade to the office staffs of the Department of Biotechnology, specially, Asit da, Biswajit da, Dipak and Raja da for their untiring cooperation and support. I am forced to break the age old saying that there are no 'Sorry' and 'Thank You' in friendship. Friends have a very special place in our lives, sharing a bond which is none others can prove to be stronger off. My friends Trishna, Amrita, Rohit, Sum on, Dipan ... have always provided me the light atmosphere which is always a bonus in a research period. Small gestures of favour counted in a big way in the form of care and support from my well wishers Soumitra da, Jishu da, Bua da, Raja da, Bublu da, Madhumita di, Tanusree di, Krishnakali, Reshmi and other members of NESPON family and specially my beloved Masimuni and Mesomoshai (Mr. Satchidananda Mukherjee ). Association with them had given me all time blessings and enough courage, with constant endeavor to carve out a niche for myself. Words are insufficient to express my feelings and my gratitude for my mother Mrs. Anima Bhowal for bringing me up and encouraging at every step of mine. I feel extremely priviledged to have a wonderful, highly caring and very supportive in -laws Mrs. Namita Saha, Mr. Amal K. Saha, Mr. Apurba Saha and Mrs. Rupa Saha to whom I offer my deepest thanks and gratitude for all round involvement in my thesis and support without which writing this thesis would not have been possible. My thanks to my sisters Nancy and Doll for taking very good care at the final stages of my compilation. My father Late Mr. Sib Sankar Bhowal , a highly cool man though worked for Army, far ahead of his time who unfortunately could not live long enough to see this thesis to fulfil his most cherished dream. Peeping his head out of this big crowd , standing apart with phenomenal qualities deserves a big applause. A big praise to someone, who is nearest, dearest and closest to my heart, a trove of intrinsic worth to cherish throughout my life, my life partner- Swarup Saha. He has been harbouring me with constant support and courage to carry on this daunting task amidst several obstacles, time and again. I thank you all. ry"-lm"- B!,..,._L .f[o.La : ']; 1J U.}J_ /).tJ e : j 5 ~ 'f)Q.c 2o II CONTENTS GENERAL INTRODUCTION 1-22 I Mineral rich acidic habitats 2 II Geological description of the sampling site: the original habitat 4 of the acidophilic autotrophic and heterotrophic strains used in the present study III Microbial ecology of AMD/ARD 5 IV Physiology of Acidophilic Chemolithoheterotrophs 8 v Sulfur Oxidizing Microorganisms 10 VI Metal resistance/ tolerance property of acidophilic II heterotrophs v Objectives of the study 13 VI References 14 CHAPTER-I 23-46 Enrichment and isolation of acid-tolerant Chemolithoheterotrophic bacteria from AMD samples of Garubathan 1.1 Introduction 24 1.2 Materials and Methods 27 1.2.1 Sampling site(s) and sampling 27 1.2.2 Enrichment of AMD samples 28 1.2.2.1 . Media 28 1.2.2.1.1 Modified9K medium containing ferrous 28 sulfate 1.2.2.1.2 Elemental sulfur medium 28 1.2.2.1.3 Thiosulfate medium 28 1.2.2.1.4 Modified DSMZ 269 medium 29 1.2.2.1.5 Modified DSMZ 269 medium containing 29 thiosulfate 1.2.2.1.6 Mixotrophic medium (MSTSY) 29 1.2.3 Preparation of solid media 29 1.2.4 Isolation and purification of acid-tolerant 30 heterotrophic bacteria 1.2.5 Screening of the acid-tolerant isolates for 31 further studies 1.2.5.1 Protein profiling 31 1.2.5.1.1 Reagents used 31 1.2.5.1.2 Preparing 12 % resolving gels 31 1.2.5.1.3 Preparing 5 % stacking gels 31 1.2.5.1.4 Pouring the slab gel 31 1.2.5.1.5 Preparation of samples 32 1.2.5.1.6 Estimation of protein in whole cells 3:2 1.2.5.1.7 Running the gel 33 1.2.5.2 Coomassie Blue Staining 33 1.2.5.2.1 Reagents used 33 1.2.5.2.2 Methods 33 1.2.5.3 Gram reaction 33 1.2.5.4 pH tolerance 34 1.2.5.5 Temperature range 34 1.2.6 Maintenance of cultures 34 1.3 Results 34 1.3.1 Sample collection 34 1.3.2 Isolation of acid-tolerant heterotrophs from A. 35 ferrooxidans culture and enrichment culture media 1.3.3 Preliminary selection of acid-tolerant heterotrophic 36 isolates based on total protein profile 1.3.4 Grouping of the selected isolates according to pH 37 tolerance, Gram nature, temperature range for growth, and nutritional versatility 1.4 Discussion 38 1.5 References 42 CHAPTER-2 47-81 Facultative sulfur Iithoautotrophy, organotrophy and general biochemical characterization of the acid-tolerant isolates 2.1 Introduction 47 2.1.1 Facultative sulfur 47 lithoautotrophy 2.1.2 Thiosulfate 48 oxidation 2.1.3 Sulfite oxidation 50 2.1.4 Organotrophy 51 2.2 Materials and Methods 52 2.2.1 Growth of acid-tolerant isolates in 52 autotrophic thiosulfate (MST) medium 2.2.2 Growth of acid-tolerant isolates in autotrophic 52 elemental sulfur medium 2.2.3 Growth of acid-tolerant isolates in mixotrophic 52 (MSTSY) medium 2.2.4 Consumption of thiosulfate by acid-tolerant 53 chemolithotrophic isolates 2.2.5 Thiosulfate and sulfite dehydrogenase assay of acid­ 53 tolerant isolates 2.2.5.1 Preparation of cell-free extract 53 2.2.5.2 Thiosulfate dehydrogenase (EC 1.8.2.2) assay 53 2.2.5.3 Sulfite dehydrogenase (EC 1.8.2.1) assay 54 2.2.6 Demonstration of organotrophic growth by the acid­ 54 tolerant isolates 2.2.6.1 Growth of acid-tolerant isolates in heterotrophic 54 (MSSY) medium 2.2.6.2 Growth of acid-tolerant isolates in R2A medium 54 2.2.7 Biochemical tests 55 2.2.7.1 Indole test 56 2.2.7.2 Methyl Red test 56 2.2.7.3 Voges-Proskauer test 56 2.2.7.4 Catalase test 57 2.2.7.5
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