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BACTERIA ASSOCIATED with INTERTIDAL SPONGE, Cinachyra Alloclada and THEIR FUNCTIONAL DIVERSITY Subina N. S. Dr. Maria-Judith

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BACTERIA ASSOCIATED with INTERTIDAL SPONGE, Cinachyra Alloclada and THEIR FUNCTIONAL DIVERSITY Subina N. S. Dr. Maria-Judith BACTERIA ASSOCIATED WITH INTERTIDAL SPONGE, Cinachyra alloclada AND THEIR FUNCTIONAL DIVERSITY A thesis submitted to Goa University for the Award of the Degree of DOCTOR OF PHILOSOPHY in MARINE SCIENCES By Subina N. S. Research Guide Dr. Maria-Judith Gonsalves Goa University, Taleigao, Goa 2018 Statement As required under the University ordinance OB-9.9 (v-vi), I state that this thesis entitled "Bacteria associated with intertidal sponge, Cinachyra alloclada and their functional diversity" is my original contribution and it has not been submitted on any previous occasion. The literature related to the problem investigated has been cited. Due acknowledgements have been made wherever facilities and suggestions have been availed of. SUBINA N. S. CSIR-National Institute of Oceanography, Goa. 4th August, 2018 Certificate This is to certify that the thesis entitled "Bacteria associated with intertidal sponge, Cinachyra alloclada and their functional diversity" submitted by Ms. Subina N. S. for the award of the degree of Doctor of Philosophy in Marine Sciences is based on original studies carried out by her under my supervision. The thesis or any part thereof has not been previously submitted for any other degree or diploma in any institution. Dr. Maria-Judith Gonsalves Research Supervisor CSIR-National Institute of Oceanography, Dona Paula, Goa 4th August, 2018 ACKNOWLEDGEMENTS I am deeply indebted to my guide, Dr. Maria-Judith Gonsalves for her encouragement and unwavering support in this work. Her positive criticism, suggestions and valuable comments has left everlasting impression on me. I also thank her for believing in me, for emotional support during adverse times, and for going the extra mile when it was necessary, in order to make this happen. I wish to express my profound thanks to Dr. Shanta Achuthankutty, for bringing me to CSIR- National Institute of Oceanography, Goa. Her initiative, constant support and help have led to the successful completion of my present investigation. Without her guidance and support, this thesis would not have been possible. I have greatly benefited from her knowledge and experience. I would like to thank the Directors of CSIR-National Institute of Oceanography, Goa for providing me with all the necessary facilities to carry out my research work. I also would like to thank CSIR for providing the infrastructure and the necessary facilities to conduct my research work. My heartfelt thanks to Dr. Achuthankutty for critical evaluation of my thesis and his valuable commens which helped to improve the thesis to the final form. I express my special thanks to my FRC members, Head of the Department, Marine Sciesnces, Dean of Life Science, Goa University (Dr. Mohandass, Prof. H. B. Menon, Prof. G. N. Nayak, Prof. C. U. Rivonkar, Prof. M. Janardhanan and Prof. Sanjeev Ghadi) for their valuable suggestions during the assessment. I feel immense pleasure in expressing my special thanks to my collegues, Feby A, Divya B, Fernandes C, Nazareth D, Kamaleson S, Sabu E, Naik A, Remya AT, Verma R, Kumar R, Linsy J, Mulla N, Shanbhag Y and Sanghodkar N for their valuable suggestions and continuous help in furtherance of my research work. My appreciation goes to Areef Sardar, for SEM analysis. I also express my gratitude to Dr. N. G. K. Karanth who directed me to research. I also thank all my teachers who encouraged me. I extend my sincere thanks to all the staffs from BOD and ITG section, and Library who directly or indirectly helped me during the completion of this work. No acknowledgement will be complete without expressing my gratitude towards my loving parents, husband and sister for their love and constant support in every walk of life. SUBINA N. S. Dedicated to Shanta ma’am (Dr. Shanta Achuthankutty) Contents CONTENTS Contents…………………………………………………………………………… i List of Tables……………………………………………………………………… vi List of Figures……………………………………………………………………... viii Chapter 1. Introduction 1. 1. General introduction……………………………………………………….. 1 1.2. Sponges: Simplest and ancient metazoan………………………………….. 1 1.3. Sponges: Reservoir of complex microbial communities………………….. 2 1.4. Sponges: Microbial fermenters……………………………………………. 4 1.5. Application…………………………………………………………. ……... 6 1.6. Sponge Cinachyra alloclada………………………………………………. 6 1.7. Objectives of the study…………………………………………………….. 7 1.8. Significance of this study………………………………………………….. 7 Chapter 2. Review of literature 2.1. Sponges…………………………………………………………………….. 9 2.1.1. Sponge habitat and their distribution………………………………………. 11 2.2. Sponge associated microorganisms………………………………………... 13 2.2.1. International scenario………………………………………………………. 16 2.2.1.1. Diversity …………………………………………………………………... 16 2.2.1.2. Functions………………………………………………………………….. 27 2.2.1.3. Biotechnological application……………………………………………… 33 Bacteria associated with intertidal sponge, Cinachyra alloclada and their functional diversity Page i Contents 2.2.2. National scenario………………………………………………………….. 37 2.2.2.1. Diversity…………………………………………………………………… 37 2.2.2.2. Function…………………………………………………………………… 38 2.2.2.3. Biotechnological application……………………………………………….39 2.3. Sponge, Cinachyra sp……………………………………………………… 40 Chapter 3. Materials and methods 3.1. Sampling site………………………………………………………………. 42 3.2. Sample collection………………………………………………………….. 42 3.3. Ambient water characteristics analysis……………………………………. 44 3.4. Microbiological analyses………………………………………………….. 45 3.4.1. Abundance of bacteria……………………………………………………... 45 3.4.1.1. Sample processing………………………………………………………… 45 3.4.1.2. Enumeration………………………………………………………………. 45 3.4.2. Diversity……………………………………………………………………..48 3.4.2.1. Culture-dependent method………………………………………………… 49 3.4.2.2. Culture-independent method……………………………………………… 50 3.4.3. Functional role of prokaryotes……………………………………………...51 3.4.3.1. Degradation and uptake of organic matter………………………………… 52 3.4.3.2. Methane oxidation………………………………………………………… 55 3.4.3.3. Nitrification……………………………………………………………….. 55 3.4.3.4. Denitrification…………………………………………………………….. 56 Bacteria associated with intertidal sponge, Cinachyra alloclada and their functional diversity Page ii Contents 3.4.3.5. Sulfate reduction………………………………………………………….. 56 3.4.3.6. Antagonistic interaction of AB against THB……………………………… 57 3.4.4. Linking bacterial phylogeny to function……………………………………. 58 3.4.5. Application of sponge-associated microbiota ………………………………58 3.4.5.1. Aquaculture wastewater treatment………………………………………… 58 3.4.5.2. Azo dye decolourization…………………………………………………... 59 3.5. Statistical analysis………………………………………………….. ……... 60 Chapter 4. Abundance and diversity of bacteria 4.1. Introduction……………………………………………………………………. 61 4.2. Result and discussion…………………………………………………………. 63 4.2.1. Water characteristics………………………………………………………… 63 4.2.2. Abundance……………………………………………………………………64 4.2.2.1 culture-independent bacterial abundance…………………………………... 64 4.2.2.2. Culture-dependent bacterial abundance…………………………………… 64 4.2.3. Diversity of bacteria…………………………………………………………. 74 4.2.3.1. Culture-dependent method………………………………………………… 74 4.2.2.2. Culture-independent method……………………………………………… 77 4.3. Conclusion…………………………………………………………………….. 85 Chapter 5. Functions of bacteria 5.1. Introduction……………………………………………………………………. 87 5.2. Results and Discussion………………………………………………………... 88 Bacteria associated with intertidal sponge, Cinachyra alloclada and their functional diversity Page iii Contents 5.2.1. Degradation of organic matter and uptake of organic and inorganic matter…88 5.2.2. Methane oxidation…………………………………………………... ……... 101 5.2.3. Nitrification…………………………………………………………………. 103 5.2.4. Denitrification………………………………………………………………. 105 5.2.5. Sulfate reduction……………………………………………………………. 107 5.2.6. Defence: Antagonistic interaction of Actinobacteria………………………. 108 5.3. Conclusion………………………………………………………………… 110 Chapter 6. Linking bacterial phylogeny to function 6.1 Introduction………………………………………………………………… 111 6.2. Results and discussion……………………………………………………... 112 6.2.1. Carbon Cycle………………………………………………………………. 118 6.2.2. Nitrogen cycle……………………………………………………………… 125 6.2.3. Sulfur cycle………………………………………………………… ……... 127 6.2.4. Defence mechanism………………………………………………………... 129 6.3. Conclusion ……………………………………………………………… 132 Chapter 7. Applications of sponge associated bacterial functions 7.1. Introduction………………………………………………………………… 133 7.2. Results and discussion……………………………………………………... 134 7.2.1. Treatment of aquaculture wastewater with sponge………………………… 134 7.2.2. Azo dye degradation by associated bacterial isolates…………………….. 139 7.3. Conclusion…………………………………………………………………..142 Bacteria associated with intertidal sponge, Cinachyra alloclada and their functional diversity Page iv Contents Chapter 8. Summary and conclusion 8.1. Introduction……………………………………………………………………. 143 8.2. The concise description of overall findings…………………………………… 147 8.2.1. Abundance…………………………………………………………………... 147 8.2.2. Diversity…………………………………………………………………….. 148 8.2.3. Functions of bacteria………………………………………………………… 149 8.2.4. Linking bacterial diversity to function……………………………………… 152 8.2.5. Application…………………………………………………………………... 154 8.3. Conclusions and future perspectives………………………………………….. 155 REFERENCES……………………………………………………………………..158 APPENDIX………………………………………………………………………... 223 Bacteria associated with intertidal sponge, Cinachyra alloclada and their functional diversity Page v List of Tables & Figures LIST OF TABLES Table 2.1. Bioactive compounds from sponge-associated bacteria Table 3.1. Tests for phenotypic identification of bacterial isolates Table 4.1. Characteristics of the ambient water Table 4.2. Abundance
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