CHARACTERIZATION AND EMULSIFYING ACTIVITIES OF A QUORUM SENSING BIOSURFACTANT PRODUCED BY A MARINE BACTERIUM A THESIS submitted by Mr. K. Abraham Peele Regd. No: 121FG01001 for the award of the degree of DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOTECHNOLOGY VIGNAN’S FOUNDATION FOR SCIENCE, TECHNOLOGY AND RESEARCH UNIVERSITY, VADLAMUDI GUNTUR – 522 213 ANDHRA PRADESH, INDIA MAY 2017 Declaration This thesis is composed of my original work, and contains no material previously published or written by another person. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, data analysis, technical procedures, editorial advice and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The Vignan's University, immediately made available for research and study. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Mr. K. Abraham Peele i THESIS CERTIFICATE This is to certify that the thesis entitled "CHARACTERIZATION AND EMULSIFYING ACTIVITIES OF A QUORUM SENSING BIOSURFACTANT PRODUCED BY A MARINE BACTERIUM" submitted by ABRAHAM PEELE KARLAPUDI to the Vignan‟s Foundation for Science, Technology and Research University, Vadlamudi, Guntur for the award of the degree of Doctor of Philosophy is a bonafide record of the research work done by him under my supervision. The contents of this thesis, in full or in parts, have not been submitted to any other Institute or University for the award of any degree or diploma. Dr. Vidya Prabhakar Kodali Research Guide Assistant Professor, Department of Biotechnology Vikrama Simhapuri University Place: Guntur Nellore Andhra Pradesh, India Date: 09 May 2017 Dr. Bharath Kumar Ravuru Research co- Guide Associate Professor, Department of Biotechnology Place: Guntur VFSTR University, Andhra Pradesh, India Date: 09 May 2017 ii ACKNOWLEDGEMENT My time as a Ph.D. student has been a special period of my life to remember, thanks to many people around me who have inspired and encouraged me to conquer this long journey. Firstly I would like to thank my university, Vignan's Foundation for Science, Technology and Research University and my supervisors, Dr. Vidya Prabhakar Kodali and Dr. R. Bharath Kumar for their patience, guidance, encouragement and trust over the course of my Ph.D. program. I am indebted to my guide, Dr. Vidya prabhakar Kodali, for accepting me as his Ph.D. student. The caring nature makes him the most wonderful person as well as guide that any scholar wishes in their Ph.D. period. I never forget his balancing behavior of guiding me scientifically, teaching me ethically and caring me more friendly. I express my sincere thanks to Prof. Ramamoorthy, Rector and incharge Vice Chancellor. I would like to especially thank my present HOD, Dr. D. Vijaya Ramu and former HOD, Prof. S. Krupanidhi who played a significant role in the success of my Ph.D. A very special thank-you goes to Ms. Kota Krishna Priya, Graduate student of Vignan's university for never ending support and extending a ready helping hand throughout my Ph.D. I take this opportunity to thank, my colleagues Mr. T.C Venkateswarulu, Mr. D. John Babu, Mrs. Indira, Mr. Ranganadh Reddy, Dr. N.S. Sampath, Dr. S. Asha, Prof. R. Venkatanadh, Mr. A. Venkatanarayana, Dr. Prakash narayan Reddy, Dr. M.S Shiva Kiran, Dept. of Biotechnology. I would also like to convey my sincere thanks to Prof. Tech Dr. Chavali Murthy Yadav, and Dr. Vidhu Kampuradh, Prof. Ravi Kumar, Prof. Avireni Srinivasulu, the members of my Ph.D. committee for giving me valuable feedbacks and keeping my research in check. I also would like to thank the non-teaching staff- Mr. Ramesh Babu, Mr. Nageswara rao, Mr. Srinivas, Mrs. Renuka Devi and Students - Rohini Krishna, Jahnavi, Lohith and Samuel. I acknowledge Department of Biotechnology (DBT, Govt. of India) for the financial support to carry out this research (Project No. BT/PR7932/AAQ/3/642/2013) and my sincere thanks to Vignan‟s University, Guntur and Vikrama Simhapuri University, Nellore, India for providing facilities. Mr. K. Abraham Peele iii ABSTRACT CHARACTERIZATION AND EMULSIFYING ACTIVITIES OF A QUORUM SENSING BIOSURFACTANT PRODUCED BY A MARINE BACTERIUM Our understanding about the ecology of hydrocarbon (oil) degrading microorganisms which are mostly bacteria, fungi have greatly enhanced in recent periods. Biosurfactants and bioemulsifers are the amphiphilic compounds that are produced extracellular or as a part of the cell membrane by bacteria. Hydrosphere consists of marine ecological environment which is more prone to oil pollution by contamination. Understanding how microorganisms degrade hydrocarbons, and thereby mitigate ecosystem damage is important to target the problem and to plan the strategy. Biosurfactants produced by biofilm-producing bacteria has a profound impact in medical biotechnology, food production, pharmaceutical, bioremediation and hydrometallurgy. Biofilms are the bacterial communities that are regulated by many signaling processes. Biofilms have the exopolysaccharide (EPS) sheath which protects the cells of the bacteria from various adverse conditions. The typical ExoPolysaccharide formed from the biofilm bacteria has the maximum carbohydrate portion and considered to be the main ingredient in the biofilm. Research suggests that amount of EPS produced by different bacteria possess different concentration and composition. Generally, the kind of bacteria which produces Exopolymeric materials such as biosurfactant, has the role in antibiotic resistance, as it offers many regulatory pathways to act against antibiotics. Natural plant extracts which contains several phenolic compounds have great role in pharmaceutical field. Due to the safety issues concerned with the natural extracts which could be a characteristic feature to become a drug, our present work deals with the screening antibiofilm agents using natural extracts and disturbing the quorum sensing signal which is responsible for the formation of the biofilm. Garlic offered a promising approach in the field of quorum sensing signal blocking mechanism. Antimicrobial compounds from the marine aquatic ecosystems delivering the promising results, bacteria live in the marine environment are deeply subjected to stress that leads to the release of various compounds in order to sustain under adverse conditions. Current work focuses on the extraction of the antimicrobial compounds from the biosurfactant producing bacteria and finding the potential bacteria as a source of novel Exopolysaccharide producing iv biofilm bacteria, in the role of oil biodegradation. Screening procedures were done to detect EPS producing biofilm bacteria. The isolate sample-M, identified as Acinetobacter species by 16S rDNA analysis, submitted to NCBI as Acinetobacter M6 strain (Accession no: KR559749). The polymer produced by Acinetobacter M6 has significant emulsification activity similar to some of the commercial emulsifiers, and also have the good surfactant activity, which appears to be a glycolipoprotein as shown by FTIR analysis. This is one of the few reports of a Acinetobacter species producing EPS with surfactant properties. The biosurfactant has shown promising results in terms of antimicrobial activity against Pseudomonas aeruginosa, therefore we can develop the drug by using pure form of biosurfactant. Bacteria produces many kinds of molecules that allow bacteria to communicate about population size, metabolic states or producing end products that initiate some activities such as bioluminescence. These molecules are generally regarded as auto inducer peptides that serves as a signal carriers. Quorum sensing reveals the fact that bacteria have the capacity to assess the number of other components they can activate, once the threshold number is reached. Acyl homoserine lactones are present mainly in gram negative bacteria and they control their own synthesis. In AHLs, the head group consists of homoserine lactones and the tail region determines the specificity of the receptor. Oligopeptide molecules are present mainly in gram positive bacteria, their synthesis is dependent on ribosomes. In the present study it is hypothesized that the production of autoinducing peptides and biofilm formation are interlinked. The small peptides which may have antimicrobial activity will also act as signals for the bacteria to form biofilm when they reach considerable cell density. Keywords: Biosurfactant, Biodegradation, Bioremediation, Hydrocarbon, Marine microbiology, Auto inducers, AHLs, Gram specific strains, oligopeptides, signaling molecules, stationary phase, quorum sensing, Exopolysaccharide (EPS) v TABLE OF CONTENTS TITLE PAGE DECLARATION i THESIS CERTIFICATE ii ACKNOWLEDGEMENT iii ABSTRACT iv TABLE OF CONTENTS vii LIST OF FIGURES ix LIST OF TABLES xi LIST OF SYMBOLS AND ABBREVIATIONS xii PATENT FILED xiii CHAPTER-I 1. Introduction 01 1.1. Hydrocarbon contamination sites 02 1.2. Surfactants 02 1.3 Biosurfactants and its classification 02 1.3.1 Glycolipids 05 1.3.2 Rhamnolipids 06 1.3.3 Trehalolipids 06 1.3.4 Sophorolipids 07 1.3.5 Lipoproteins and Lipopeptides 07 1.3.6 Lichenysin 08 1.3.7 Phospholipids and Fatty acids 08 1.3.8 Polymeric biosurfactants 08 1.4. Properties of biosurfactants 09 1.4.1 Surface and interface activity 09 1.4.2 Temperature, pH and ionic strength tolerance 09 1.4.3 Biodegradability 10 1.5 Biofilm 10 1.6 Thesis objectives 12 1.7 Thesis organizations 12 CHAPTER -II REVIEW OF LITERATURE 2.1 Introduction 13 2.2 Microbial enhanced oil recovery (MEOR) 18 2.3 Oil Contamination and Remediation 19 2.4 Petroleum hydrocarbon degradation 21 2.5 Antibiotic Degradation 22 2.6 Case research 23 2.7 Biofilm formation 25 2.8 Purification procedures 26 2.9 Identification and Structural analysis 26 2.10 Biological activity of surfactants 27 2.10.1 Inflammation 27 2.10.2 Cyclooxygenases and Lipoxygenases 27 2.11 Motivation 28 vi CHAPTER-III EMULSIFICATION POTENTIAL OF BIOSURFACTANT PRODUCED FROM ACINETOBACTER M6 3.1 Introduction 29 3.2 Methodology and Research Design 30 3.2.1 Isolation and screening of biosurfactant producing bacteria and Biofilm Assay 30 3.2.2 Estimation of crude oil degradation 30 3.2.3 Screening of hydrocarbon degraders by 2,6-DCPIP oxidation test 31 3.2.4 Biochemical characterization and Hemolysis test 31 3.2.5 16S rDNA sequence analysis 31 3.2.6 Biofilm assay and EPS estimation 31 3.2.7 Estimation of Biomass 32 3.2.8 Effect of pH, temperature, carbon, nitrogen and sodium chloride on biosurfactant Production 32 3.2.9 Surface tension and the Critical Micelle Concentration 33 3.2.10 Emulsifying potential of the biosurfactant 33 3.2.11 Staining and Visualization of EPS with fluorescently labeled lectins 33 3.2.12 Gel Filtration Chromatography, TLC and FTIR analysis 33 3.3 Results and Discussion 34 3.3.1 Isolation and screening of iosurfactant producer 35 3.3.2 16S rDNA sequence analysis 36 3.3.3 Staining and visualization of biofilm EPS with fluorescently labeled lectins 38 3.3.4 Biosurfactant production and quantification 38 3.3.5 Effect of Media, pH, temperature, carbon, nitrogen and sodium chloride on biosurfactant Production 39 3.3.6 Surface tension and the Critical Micelle Concentration 47 3.3.7 Emulsifying potential of biosurfactant 48 3.3.8 Gel filtration chromatography, TLC and FTIR 51 3.4 Summary 54 CHAPTER-IV ANTI- INFLAMMATORY STUDIES OF A BIOSURFACTANT 4.1 Introduction 55 4.2 MethodologyandResearchDesign 56 4.2.1 Extraction,Quantification of biosurfactantfrom Acinetobacter M6 56 4.2.2 Protein Purification using 2D cleanup 56 4.2.3 Iso-electrofocusing and 2 Dimensional SDS-PAGE 57 4.2.4 Coomassie Brilliant Blue (CBB) and Periodic Acid Schiff‟s (PAS) staining 57 4.2.5 Protein in gel-digestion and identification 57 4.3 Anti-inflammation studies 58 4.3.1 Solvent extractions of biosurfactant fractions 58 4.3.2 Inhibition of 5-LOX activity by biosurfactant 58 4.3.3 Inhibiton of Cyclooxygenase (COX-1 & COX-2) activity 59 4.4 Results and Discussion 60 4.4.1 Biofilm formation and quantification of Biosurfactant 60 4.4.2 SDS-PAGE 60 4.4.3 MALDI-TOF analysis of peptides 61 4.5 Anti-inflammation studies 73 4.5.1 Inhibition of 5-LOX activity by crude, partially purified pellets of biosurfactant 73 vii 4.5.2 Inhibiton of Cyclooxygenase (COX-2) activity 74 4.5.3 Inhibiton of Cyclooxygenase (COX-1) activity 74 4.6 Summary 75 CHAPTER-V QUORUM SENSING STUDIES OF BIOSURFACTANT PRODUCTION 5.1 Introduction 77 5.1.1 Quorum sensing in bacteria 79 5.2 Materials and methods 80 5.2.1 Biosurfactant extraction 80 5.2.2 Biofilm formation and SEM 80 5.2.3 Inhibition studies of the biofilm and biosurfactant production using plant extracts 81 5.2.4 Study of the effect of plant extracts on biosurfactant production 81 5.2.5 Anti susceptibility test of the Acinetobacter M6 81 5.3 Quorum sensing studies 81 5.3.1 Extraction of Ginger and Garlic Juice 81 5.3.2 N-Acyl Homoserine Lactone peptide extraction from Acinetobacter M6 82 5.3.3 The Antimicrobial Activity of biosurfactant 82 5.3.4 DPPH radical scavenging assay 82 5.4 RESULTS AND DISCUSSION 82 5.4.1 Effect of plant extracts on Biofilm and Biosurfactant Production 82 5.4.2 Anti susceptibility test of the Acinetobacter M6 83 5.5 Quorum sensing studies 84 5.5.1 Estimation of growth profiles in broth and supernatant inoculated cultures 84 5.5.2 Biofilm formation and SEM 85 5.5.3 Quorum sensing studies 86 5.5.4 The Antimicrobial Activity of biosurfactant 88 5.5.5 DPPH radical scavenging assay 89 5.6 Summary 90 CHAPTER-VI CONCLUSIONS AND SCOPE OF FUTURE WORK 6.1 Conclusions 93 6.2 Scope of Future work 94 viii