Bioprospecting of antibacterial metabolites in seaweed associated bacterial flora along the southeast coast of India Thesis submitted to COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in MARINE SCIENCES By BINI THILAKAN (Reg. No.3782) CENTRAL MARINE FISHERIES RESEARCH INSTITUTE (Indian council of Agricultural Research) Post Box No. 1603, Cochin-682018, INDIA October 2015 I do hereby declare that the thesis entitled “Bioprospecting of antibacterial metabolites in seaweed associated bacterial flora along the southeast coast of India” is the authentic and bonafide record of the research work carried out by me under the guidance of Dr. Kajal Chakraborty, Senior Scientist, Central Marine Fisheries Research Institute, Cochin, in partial fulfilment for the award of Ph.D. degree under the Faculty of Marine Sciences of Cochin University of Science and Technology, Cochin and no part thereof has been previously formed the basis for the award of any degree, diploma, associate ship, fellowship or other similar titles or recognition. Place: Cochin Date: 16/10/2015 BINI THILAKAN Dedicated to my family On this occasion of completing my doctoral degree dissertation, I realize that this work was not completed in a vacuum. I am thankful to many people who have helped me through the completion of this dissertation. Without thanking them my work will be incomplete. The first and foremost I wish to thank my supervisor, Dr.Kajal Chakraborty, Senior Scientist, Marine Biotechnology Division, CMFRI. who has been supportive since the days i joined CMFRI as a project assistant. He helped me come up with the thesis topic and guided me through the rough road to finish this thesis. Thanks to him for supporting me academically over almost a year of development and for the encouragement and the freedom i needed to move on. I feel extremely privileged to thank Dr. A. Gopalakrishnan, Director, Central Marine Fisheries Research Institute for being kind enough to extend all the necessary facilities and support required to carry out my research work. I record my deep sense of respect and gratitude to him for providing me with the absolute freedom to successfully complete this thesis. I extend my deep sense of gratitude and respect to Dr. G. Syda Rao, former Director of Central Marine Fisheries Research Institute for being kind enough to permit me to register for the Ph.D. programme and providing me with all the necessary facilities required to carry out this work. I express my heartfelt gratitude to Dr. Boby Ignatius, Principal Scientist, HRD Cell for kindly facilitating my Ph.D. programme. I feel privileged to place on record my sincere gratitude to Dr. P.C. Thomas for facilitating my doctoral work during his tenure as Scientist-in-Charge, HRD cell of CMFRI. I express my deep sense of gratitude to Dr. P. Vijayagopal, HOD, Marine Biotechnology Division, CMFRI for his consistent help, guidance and encouragement during the research work. I extend my deep sense of gratitude and respect to Dr. K.K.Vijayan, former HOD, Marine Biotechnology Division, CMFRI for his consistent help, guidance, subjective criticism and encouragement during the research work. I extend my sincere thanks to Dr. Rekhadevi Chakraborty, Mr. N.K Sanil, Dr. V. Srinivasa Raghavan, Dr. Pradeep M.A and Dr. Sandhya Sukumaran for all the encouragement and support. I wish to express my heartful thanks to Dr. M.P. Paulton (Senior Technical Officer), Mr. Nandakumar Rao, Mr. K.K Surendran (Technical officers, CMFRI), and Mrs. P. Vineetha, of Marine Biotechnology Division, CMFRI for their cooperation and help. I express my deepest sense of gratitude to Mr. Edwin Joseph, OIC library, library support staff, the canteen, security personnel and employees of CMFRI, Cochin, for their sincere help and cooperation extended during the tenure. I acknowledge the staffs of SAIF, IIT Chennai and staffs of STIC, Cochin University of Science and Technology (CUSAT) for their help and support extended towards the spectroscopic studies. I express my heart felt gratitude to my colleagues Dr. N. K. Praveen, Mr. Deepu Joseph and Mrs. Selsa Chakkalakkal for bouncing ideas with me. I am grateful to Vamsi Krishna Raola for his help extended towards compiling my spectroscopic studies. I express my deep sense of gratitude to Dr.Anju Antony, Dr. Vikas P.A and Mrs. Vidya Susan Philip for their support and help. I remember Mr. Jackson K.V, Mrs. Anijo, Mr.Neil, Mrs. Anusree, , Mr. Reynold Peter, Mr. Renjith Kumar, Mr. Sajesh Kumar, Mrs. Preetha, Mrs. Suja, Mr. Subin, Mr. Raja, Mrs Sreedevi, Mrs Keerthi, Mrs Githa, Mrs Sajeela and all my junior research scholars of MBTD for their timely help. I also thank Central Marine Fisheries Research institute and organization, ICAR; under which I completed my research. Last but not the least I thank my entire family for their support and encouragement. I express my love towards my little ones Avishai and Upasana who sacrificed the most during my research work. I am indebted to my loving husband Mr. Sreenesh N.S for his care, cooperation, endless patience and encouragement throughout the period of my work. I remember my heavenly father MR. P.K. Thilakan and mother Baby K.P with profound sense of gratitude whose selfless sacrifice and their great efforts with pain and tears and unceasing prayers has enabled me to successfully complete the research work. My special gratitude is due to my Sisters Nidhi, Nila and brother-in- law ‘Sreejith N.S’ for their loving support. I am forever indebted to my parents-in-law for their help and support which made my task much easier. Finally, I thank all those who have helped me directly or indirectly in the successful completion of my thesis. Above all I humbly bow before the almighty God for showering his blessings upon me and giving me the strength, wisdom, health and luck to accomplish this endeavor. Abbreviation 1HNMR - Proton Nuclear Magnetic Resonance BLAST - Basic local alignment search tool COSY - Correlation Spectroscopy DEPT - Distortionless Enhancement By Polarization Transfer DNA - deoxyribonucleic acid EMBL - European Molecular Biology Laboratory EtOAc - Ethyl Acetate FAME - Fatty Acid Methyl Esters HMBC - Heteronuclear Multiple Bond Correlation HPLC - High-Performance Liquid Chromatography HRMS - High Resolution Mass Spectroscopy HSQC - Heteronuclear Single Quantum Coherence Min - minutes NCBI - National centre for biotechnology information NMR - Nuclear Magnetic Resonance NOESY - Nuclear Overhauser Effect Spectroscopy NRPS - Nonribosomal peptide synthetase PCR - polymeras chain reaction PKS - Polyketide synthetase P-TLC - Preparative Thin Layer Chromatography Rf - Retardation Factor Rt - Retention Time TBE - Tris boric acid Ethylene diamine tetra acetic acid TLC - Thin Layer Chromatography UV-VIS - Ultra Violet-Visible NA - Nutrient agar ZMA - Zobell Marine agar SWA - Sea water agar Contents Page No: Chapter 1 Introduction 1-13 1.1 Background 1.2 Seaweed resistance to microbial attack 1.3 The symbiotic role of marine microbes on living surfaces 1.4 Bacterial communities coexisting with the seaweeds as a source of novel natural compounds 1.5 Functional genes associated with the seaweed-associated bacteria and their relation to the bioactivity 1.6 Objectives of the proposed work 1.7 Thesis outline Chapter 2 Review of literature 14-47 2.1 Background 2.2 Marine Microorganisms as a Source of Bioactive Metabolites 2.3 Microbial natural products 2.4 Natural product derived drug molecules 2.5 Bioactivities of molecules from microbial flora 2.5.1 Immunosuppresant molecules from marine microbial flora 2.5.2 Antitumor drugs from microbial sources 2.5.3 Microbial products in agriculture 2.5.4 Aquaculture grade chemicals from marine microbes 2.6 New molecules from marine bacteria as a solution towards multiresistant antibiotic and drug molecules 2.7 Symbiotic role of marine microbes and bioactive compound production 2.8 Why seaweed associated bacteria? 2.8.1 Seaweed associated bioactive bacterial isolates 2.8.2 Protective role of seaweed associated bacteria against fouling 2.8.3 Seaweed associated bacteria against fish pathogens 2.9 Seaweed associated bacterial as a source of antimicrobial compounds 2.10 Diversity of seaweed associated antibiotic producers 2.11 Antibiotic Compounds from Bacillus: Why are they so amazing? 2.12 Functional genes related to the antimicrobial activity of the seaweed associated bacteria 2.13 Conclusions Chapter 3 Isolation of antagonistic bacterial flora associated with seaweeds 48-81 3.1 Background 3.2 Materials and Methods 3.2.1 Seaweed sample collection 3.2.2 Isolation of Seaweed-Associated Bacteria 3.2.3 Purification of Seaweed-Associated Bacterial isolates 3.2.3.1 The streak-plate method for isolation of pure cultures 3.2.4 Pathogenic test organisms used for the study 3.2.4.1 Vibrio parahaemolyticus 3.2.4.1.1 Vibrio parahaemolyticus (ATCC® 17802™) 3.2.4.1.2 Vibrio parahaemolyticus MTCC451 3.2.4.2 Vibrio vulnificus MTCC1145 3.2.4.3 Vibrio harveyi 3.2.4.4 Aeromonas hydrophilla 3.2.4.5 Vibrio alginolyticus 3.2.4.6 Vibrio angullarum 3.2.5 Screening of isolated strains for their antibacterial activity against the pathogens 3.2.6 Preservation of isolated strains with antibacterial activity against the tested pathogens 3.2.6.1 Serial sub culture on agar slants 3.2.6.2 Oil sealing 3.2.6.3 Storage at low temperature 3.2.6.4 Preservation by Lyophillization 3.2.6.5 Preservation using Seaweed Extract Agar 3.2.7 Identification of isolated strains with antibacterial activity 3.2.7.1 Biochemical identification 3.2.7.2 Molecular identification based on 16S rrna genes 3.2.7.2.1 DNA isolation 3.2.7. 2.2 PCR 3.2.7.2.3 BLAST 3.2.7.2.4 Phylogenetic tree construction 3.2.7.2.5 Nucleotide Accession numbers 3.2.7.3 Chemotaxonomy based on analysis of fattyacid profiles 3.2.7.