Flinders University Department of Medical Biotechnology School of Medicine --------------------------------------- Identification and Characterization of Endophytic Actinobacteria Isolated from Plants and Sponges Alhnouf Alqurashi Department of Medical Biotechnology School of Medicine, Faculty of Health Sciences Flinders University A thesis submitted for the degree of Masters of Biotechnology Studies 2016 _________________________________________________________ Table of Contents CHAPTER ONE: INTRODUCTION ......................................................................... 1 1. General overview .................................................................................................................... 1 1.1 General Aspects of Actinobacteria .............................................................................. 2 1.1.1 Morphological and genetics features of Actinobacteria ......................................... 2 1.1.2 Classification of Actinobacteria ............................................................................. 2 1.1.3 Activities of Actinobacteria in the natural habitat ................................................... 2 1.1.4 Endophytic Actinobacteria ..................................................................................... 3 1.2 Applications of Actinobacteria ................................................................................... 4 1.2.1 Industrial Application of Actinobacteria ................................................................ 4 1.2.2 Agricultural Application of Actinobacteria ............................................................ 4 1.3 General Aspects of Secondary Metabolites ............................................................... 5 1.3.1 Description of Secondary Metabolites .................................................................. 5 1.3.2 Functions of Secondary Metabolites ..................................................................... 6 1.3.3 Screening of Secondary Metabolites ..................................................................... 8 1.4 Native Australian Plants ............................................................................................. 9 1.4.1 Application and Innovation ........................................................................................ 9 1.5 Marine Actinobacteria .............................................................................................. 11 1.5.1 Applications and Innovation ..................................................................................... 11 1.6 General Aspects of Antibiotics ................................................................................. 14 1.6.1 History of Antibiotics ............................................................................................... 14 1.6.2 Antibiotics Mechanisms of Action ........................................................................... 15 1.6.3 Antibiotic Resistance in Bacteria ............................................................................. 16 1.7 Detection of Secondary Metabolites ......................................................................... 17 1.7.1 Thin Layer Chromatography (TLC) .......................................................................... 19 1.7.2 High Pressure Liquid Chromatography (HPLC) ...................................................... 19 CHAPTER TWO: MATERIALS AND METHODS ....................................... 21 2.1 Microorganisms Used ................................................................................................ 21 2.1.1 Actinobacteria Used ............................................................................................. 21 2.1.2 Test Organisms ..................................................................................................... 21 2.2 Morphological Characterization of Actinobacteria ............................................... 22 2.3 Extraction and Processing of Actinobacteria Culture Extracts ............................ 22 2.3.1 Actinobacteria Agar Metabolites Extraction ........................................................ 23 2.3.2 Small-Scale Actinobacteria Fermentation Metabolites Extraction ...................... 23 2.3.3 Large-Scale Production and Recovery of Antimicrobial Metabolites .................. 24 2.4 Concentration of Extracts ......................................................................................... 24 2.5 Bioassay of Secondary Metabolites .......................................................................... 25 2.5.1 Plug Type Bioassay .............................................................................................. 25 2.5.2Well Type Bioassay ............................................................................................... 26 2.6 Fermentation of Actinobacteria ............................................................................... 26 ii 2.6.1 Small-Scale Submerged Shake-Flask Fermentation ............................................. 26 2.6.2 Large-Scale Submerged Shake-Flask Fermentation ............................................. 28 2.6.3 Solid-State Fermentation ...................................................................................... 29 2.7 Detection of The Compound ...................................................................................... 29 2.7.1 Thin Layer Chromatography (TLC) ...................................................................... 29 2.7.2 Bioautography ...................................................................................................... 30 2.7.3 High Pressure Liquid Chromatography (HPLC) ................................................... 30 2.8 Identification of Endophytic Actinobacteria Based on 16S rRNA Gene Sequencing ............................................................................................. 30 2.8.1 Extraction of Bacterial DNA ................................................................................ 30 2.8.2 Polymerase Chain Reaction (PCR) ........................................................................ 31 2.8.3 Purification of PCR Product ................................................................................. 32 2.8.4 Agarose Gel Electrophoresis ................................................................................ 32 2.8.5 16S rRNA Gene Sequencing ................................................................................ 33 2.8.6 Phylogenetic Analysis .......................................................................................... 33 CHAPTER THREE: RESULTS ........................................................................................ 34 3.1 Morphological Characteristic of Actinobacteria Cultures ........................ 34 3.2 Primary Antibiotic Assay ......................................................................................... 39 3.3 Fermentation .............................................................................................................. 40 3.3.1 Submerged Shake-Flask Fermentation ................................................................ 40 3.3.2 Solid State Fermentation ..................................................................................... 41 3.4 Antibacterial Activity by Submerged Fermentation .............................................. 42 3.4.1 Antibacterial Activity Against S.aureus .............................................................. 42 3.4.2Antibacterial Activity Against M.luteus ............................................................... 42 3.5 Secondary Metabolites Detection ............................................................................. 43 3.5.1 Thin Layer Chromatography (TLC) ..................................................................... 43 3.5.2 Bioautography ..................................................................................................... 49 3.5.3 High Pressure Liquid Chromatography (HPLC) .................................................. 49 3.6 Identification of Endophytic Actinobacteria .......................................................... 52 3.6.1 16S rRNA Sequencing ......................................................................................... 52 3.6.2 Phylogenetic Tree ................................................................................................ 53 CHAPTER FOUR: DISCUSION ..................................................................................... 55 4.1 16S rRNA Sequencing ................................................................................................. 55 4.2 Fermentation ................................................................................................................ 56 4.3 Bioassay ........................................................................................................................ 57 4.4 Detection of Secondary Metabolites .......................................................................... 58 iii CHAPTER FIVE: CONCLUSION AND FUTURE DIRECTION .......................... 59 5.1 Conclusions ................................................................................................................. 59 5.2 Future Directions ......................................................................................................... 60 REFERENCES ................................................................................................................................. 61 iv ACKNOWLEDGMENT Apart