Role of Metopus es in the anaerobic degradation of organic matter and biomethanation process Thesis submitted to the University of Kerala for award of the degree of Doctor of Philosophy in Microbiology By Nimi Narayanan Under the Supervision of Dr. V. B. Manilal Process Engineering and Environmental Technology Division, National Institute for Interdisciplinary Science and Technology (NIIST), CSIR, Thiruvananthapuram, Kerala, INDIA - 695019 2011 To my Family DECLARATION I hereby declare that the work presented in this thesis is based on the original work done by me under the guidance of Dr. V. B. Manilal, Principal Scientist, Process Engineering and Environmental Technology Division, National Institute for Interdisciplinary Science and Technology, and that no part of this has been included in any other thesis submitted previously for the award of any degree. Nimi Narayanan Acknowledgement It is a great pleasure to express my sincere gratitude and sense of appreciation to my research guide, Dr. V.B. Manilal, Principal Scientist, Environmental Technology, NIIST, Trivandrum, for his constant encouragement, enthusiastic support and valuable guidance throughout the period of study. I am indebted to him for giving me the ample freedom to do the work and express my ideas during this period. I am grateful to Dr. Ajit Haridas, Scientist-in-charge, Environmental Technology, NIIST for his valuable suggestions and constructive criticism during my tenure. It is an honor for me to thank the present Director, Dr. Suresh Das and the former directors of NIIST, Trivandrum for providing the necessary infrastructural facilities for the successful completion of work. I would like to thank Council of Scientific and Industrial Research, New Delhi, for the research fellowship. I am extremely thankful to Dr. B. Krishnakumar, Scientist, Environmental Technology, for his valuable suggestions and constant help during the period. I would like to thank Dr. Rugmini Sukumar, Dr.Anoop Krishnan, Mr. J. Ansari, Mrs. Vijaya Prasad and Mr. K.S. Raot for their valuable suggestions and timely help throughout the work. I would also like to thank Mr. Chandran for helping with SEM work. I express my sincere gratitude to Mr. Shajikumar for his constant encouragement and valuable advices during my tough time. His caring attitude will be cherished by me through all walks of the life. I am indebted to Miss Anupama for her help in FISH analysis and moral support during my tough time. Her sisterly affection would be cherished by me throughout the life. I owe my sincere thanks to my friends, Asha Poorna and Alan Sheeja for their constant moral support and encouragement during the entire period. It is a great pleasure to convey my heartfelt thanks to my friends, Meena, Abhilash, Priya, Sumi, Nicemol, Subi, Sankar, Hima, Shyju, Faizal, Pratheesh and all friends of NIIST for their support and help throughout the work. With deep sense of gratitude I remember all of my teachers from school and colleges for motivation, inspiration and whole hearted help. Above all, I am deeply indebted to my loving father, late mother, sister, brother, uncle and all family members for their valuable care, support, encouragement and prayers. My husband Rajesh holds a special place in making this thesis through his constant support and unfailing endurance. Last but not the least; I acknowledge God almighty for being always with me Nimi Narayanan CONTENTS Page Number Table of Contents i List of publications ix Abbreviations x List of Figures xi List of Tables xv Abstract xvii Tables of Contents CHAPTER 1 ............................................................................................ 1 Introduction .......................................................................................... 1 1.1 Biomethanation process ............................................................................... 1 1.1.1 Hydrolysis .................................................................................................................. 3 1.1.2 Acidogenesis .............................................................................................................. 3 1.1.3 Acetogenesis ............................................................................................................. 4 1.1.4 Methanogenesis ........................................................................................................ 4 1.2 Biomethanation process for wastewater treatment ...................................... 5 1.3 Microbial communities in biomethanation process for wastewater treatment .......................................................................................................................... 7 1.3.1 Hydrolytic bacteria .................................................................................................... 7 1.3.2 Acidogenic bacteria ................................................................................................... 8 1.3.3 Acetogenic bacteria ................................................................................................... 9 1.3.4 Methanogens .......................................................................................................... 10 1.4 Protozoa in biomethanation processes ........................................................14 1.5 Protozoa .....................................................................................................15 i 1.5.1 Habitats ................................................................................................................... 15 1.5.2 Morphology ............................................................................................................. 16 1.5.3 Nutrition .................................................................................................................. 17 1.5.4 Formation of cyst .................................................................................................... 17 1.5.5 Reproduction ........................................................................................................... 18 1.5.6 Classification of protozoa ........................................................................................ 18 1.6 Broad functional groups of free living protozoa ...........................................23 1.6.1 Ciliates ..................................................................................................................... 23 1.6.2 Heterotrophic flagellates ........................................................................................ 25 1.6.3 Sarcodines ............................................................................................................... 26 1.7 Anaerobic protozoa .....................................................................................26 1.7.1 The metabolism in anaerobic protozoa .................................................................. 29 1.7.2 The hydrogenosomes .............................................................................................. 31 1.7.3 Symbiotic association with prokaryotes ................................................................. 34 1.7.3.1 Phototrophic endosymbiotic bacteria in anaerobic protozoa ......................... 34 1.7.3.2 Methanogenic endosymbiotic bacteria in anaerobic protozoa ...................... 35 1.7.3.3 Ectosymbiotic sulfate reducing bacteria in anaerobic protozoa ..................... 36 1.8 Metopus ......................................................................................................37 1.8.1 Classification of Metopus ........................................................................................ 38 1.8.2 General morphology of Metopus ............................................................................ 38 1.8.3 Culturing of Metopus .............................................................................................. 41 1.8.4 Endosymbiotic interactions in Metopus ................................................................. 42 1.8.5 Organic matter degradation by Metopus ............................................................... 43 1.9 The objectives and relevance of the present study .......................................44 CHAPTER 2 .......................................................................................... 46 Isolation, culturing and identification of Metopus sp. from the anaerobic reactor ................................................................................ 46 PART A ..............................................................................................................46 ii Isolation of Metopus sp. from anaerobic reactor and development of monoculture .....................................................................................................46 2.1.1 Introduction ............................................................................................................ 46 2.1.2 Materials and methods ........................................................................................... 48 2.1.2.1 Population dynamics of Metopus sp. in anaerobic UASB reactor ................... 48 2.1.2.2 Microscopic observations ................................................................................ 49 2.1.2.2a Fixation of Metopus sp. ................................................................................. 49 2.1.2.2b Enumeration of Metopus sp. ......................................................................... 50 2.1.2.3 Isolation of Metopus sp. from reactor samples ..............................................