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Front Matter Construction of metagenomic clone library for microbial diversity assessment in an electronic waste polluted site and biosorption of heavy metals by isolated organisms Thesis Submitted to Bharathidasan University for the Award of the Degree of Doctor of Philosophy in Environmental Biotechnology By Ramasamy RAJESHKUMAR, M.Sc., M.Phil., PGDBI Under the Supervision of Dr. K. THAMARAISELVI Assistant Professor Department of Environmental Biotechnology Bharathidasan University Tiruchirappalli - 620 024 Tamilnadu, India December 2011 Department of Environmental Biotechnology School of Environmental Sciences BHARATHIDASAN Tiruchirappalli – 620 024. UNIVERSITY Tamilnadu, India. Phone : +91-431-2407088 Fax : +91-431-2407045 Dr. K. Thamaraiselvi Email : [email protected] Assistant Professor [email protected] CERTIFICATE I hereby certify that the thesis entitled “Construction of metagenomic clone library for microbial diversity assessment in an electronic waste polluted site and biosorption of heavy metals by isolated organisms” submitted to Bharathidasan University, Tiruchirappalli, Tamilnadu, India, for the award of Doctor of Philosophy in Environmental Biotechnology is an authentic record of original work carried out by Mr. R. Rajesh kumar, MSc., MPhil., PGDBI., under my supervision at Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamilnadu, India. I further certify that no part of the thesis has been submitted elsewhere for the award of any other degree, diploma or associate-ship of any other University. K. Thamaraiselvi Declaration I do hereby declare that the research work on “Construction of metagenomic clone library for microbial diversity assessment in an electronic waste polluted site and biosorption of heavy metals by isolated organisms” has been originally carried out by me in the Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamilnadu, India and part of the work (Metagenomics) carried out in the Environmental Genomics Divison, National Environmental Engineering Research Institute (NEERI - CSIR), Nagpur, Maharashtra, India. I further declare that this work has not been submitted elsewhere for any other degree or other similar titles. To the best of my knowledge, this is the first metagenomic study for the assessment of microbial diversity in an e-waste recycling facility polluted surface soil. And this is the first study on employing of metal tolerant bacteria isolated from an e-waste recycling facility polluted surface soil for the removal of heavy metals. R. Rajeshkumar Tiruchirappalli Acknowledgement I avail this opportunity to express my heartfelt thanks to helping hands behind this work. It is still great at this juncture to recall all the faces and spirits in the form of faculties, friends, near and dear ones. I would consider this work nothing more than incomplete without attending to the task of acknowledging the overwhelming help I received during this endeavor. I express my thanks to research supervisor Dr. K. Thamaraiselvi, Assistant Professor, Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamilnadu, India At the very outset, I feel inadequacy of words to express my profound indebtedness and deep sense of gratitude to my mentor, School chair and Head of the Department Dr. M. Krishnan, Professor, Department of Environmental Biotechnology, Bharathidasan University without whose appreciation, valuable advice and constant encouragement, this task would not have been accomplished. His interaction with students, freeness, helping nature, freedom to work and communication style really appreciable. He took care of my personnel problems also and helped me whenever I was in trouble. Special thanks to Dr. Hemant J. Purohit, Scientist G and Head, Environmental Genomics Divison, NEERI(CSIR), Nagpur, Maharashtra, India for considering me as a visiting researcher to carry out metagenomics study under his valuable guidance, kind cooperation and providing all the facilities to work in his laboratory. I extend my thanks to Dr. Atya Kapley, Scientist, for her valuable guidance and critical suggestion during the study. I acknowledge with sincere thanks to doctoral committee member Dr. Vasanthi Nachiappan, Associate Professor and Head, Department of Biochemistry, Bharathidasan University, Tiruchirappalli, Tamilnadu, India for her constant help, guidance, co-operation throughout this Ph.D program. I am overwhelmed with sincere feelings of indebtedness to Dr. M. Ravichandran, Professor and Head, Department of Environmental Management, Bharathidasan University, Thiruchirappalli, Tamilnadu, India for his help and support throughout this Ph.D program. My thanks to Dr. R. Babu Rajendiran, Associate Professor, Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamilnadu, India for encouragement during the period of investigation. I am very much inspired by his hardworking nature, affection towards the department and students. I owe this opportunity to thank Dr. S. Achiraman, Assistant Professor, Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamilnadu, India for his help, advice and constructive criticism. I express my profound sense of gratitude to Dr. M. Vasanthy and Dr. T. Sivasudha, Assistant Professors, Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamilnadu, India for their fruitful guidelines and advice during this Ph.D program. I feel grateful to University Grants Commission, Ministry of Human Resource Development, Government of India, New Delhi for awarding me Rajiv Gandhi National Research Fellowship and providing me financial assistance. My untold sense of gratitude and profound indebtedness to my beloved parents Mr. P. Ramasamy and Mrs. Jelina Ramasamy without their blessings I would not have grown to this level, who are always with me and have taken a lot of pain to bring me up to this level. I would like to express my sincere love and thanks to my younger sister Dr. R. Priyadarshini, Physician Assistant, Meenakshi Mission Hospital, Madurai, Tamilnadu, India for her continuous encouragement, helpful comments and suggestions in my life. No words in this mortal world can suffice to express my feelings towards my uncles Er. K. Gandhi, BE, PGDCS, Pudhur M. Anbazhagan, Kavignar and Writer, and their family for their intangible encouragement, love and affection towards me. I am also thankful to Mr. Murugesan and Mr. K. Saravanan for their care, love and affection towards me. I am overwhelmed with lot of affection showered on me by all my family members, relatives and neighbors. I express my thanks and indebtedness to my beloved lab mates and emerging Environmental Biotechnologists Ms. Valsala, Ms. Poornima Priyadarshini, Mr. Prakash and Ms. Elavarasi for their help and encouragement during this investigation. I would like to thank my friends from other departments Mr. T. Manikandan, Mr. S. P. Manivannan, Ms. Kavitha, Ms. Sugunadevi, Mr. Ganesan, Mr. Karthikeyan and others for the happiest moments they provided to me and for their constant encouragement which helped to sustain my interest and thereby accomplishing this task. I would like to express my thanks to Mr. R. Rajesh Pal, Ms. Leena Agarwal and other Researchers, Environmental Genomics Division, NEERI (CSIR), Nagpur, Maharashtra, India. I also express my thanks to the non-teaching staff Mr. Tharasingh, Mr. Muralidharan and Mr. Tirupathi, Department of Environmental Biotechnology and Mr. Logesh, Finance Section, Bharathidasan University, Thiruchirappalli, Tamilnadu, India for their generous and timely help. Rajeshkumar. R Affectionately Dedicated to My Beloved Parents and Research Community CONTENTS Chapter No. Title Page No. I General Introduction 1 II Review of Literature 9 III Materials and Methods 60 IV Results and Discussion 77 V Summary and Conclusion 129 References i - xxxvi Appendices LIST OF ABBREVIATIONS % Percentage °C celcius µl microlitre bp base pairs bp basepair CFU Colony Forming Unit g grams h hours H2SO4 Sulphuric Acid L litres L-1 per litre LB Luria Bertani M molar mg milligram min minutes ml millilitre N normality ng nano gram nm nano metre OD optical density pH concentration of hydrogen ions ppm parts per million rpm rotation per minute sec seconds TFTC too few to count TNTC too numerous to count V volts μg microgram μM Micro molar Chapter I 1. Introduction For much of the last century, microbiologists have been aware that the identity of only a tiny fraction of the inhabitants of the microscopic landscape. While most people are very familiar with the diversity of life in the plant and animal kingdoms, few actually realize the vast amounts of variability present in the bacterial populations. Microorganisms represent the richest repertoire of molecular and chemical diversity in nature as they underlie basic ecosystem processes. The current inventory of the world’s biodiversity is very incomplete and that of viruses, microorganisms and invertebrates is especially deficient. Scientists have identified about 1.7 million living species on our planet. Studies indicate that the 5,000 identified species of prokaryotes represent only 1 to 10% of all bacterial species, therefore we have only a small idea of our true microbial diversity (Stanley, 2002). Microorganisms constitute a huge and almost unexplained reservoir of resources likely to provide innovative applications useful to man. Microorganisms have been evolving
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