PRODUCTION OF NATURAL PIGMENT WITH ANTIMICROBIAL 2.4 Functions of bacterial natural pigments 33 2.4.1

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    PRODUCTION OF NATURAL PIGMENT WITH

    ANTIMICROBIAL ACTIVITY FROM A MARINE

    BACTERIUM,

    Pseudoalteromonas rubra BF1A IBRL

    by

    AZLINAH BINTI MOHD SULAIMAN

    Thesis submitted in fulfilment of the requirements

    for the degree of

    Doctor of Philosophy

    February 2016

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    ACKNOWLEDGEMENT

    With deep gratitude, I would like to express my appreciation to my

    honourable supervisor Professor Darah Ibrahim for her guidance, patience,

    encouragements and advices given to me in completing this research work. She has

    been my supervisor since in year 2005, where I began my Masters study, and I am

    glad to work under her supervision. Her guidance and her trust on me had

    encouraged me so much until I completed this study. Thank you very much to her.

    I am also thankful to my co-supervisor, Dr. Wan Norhana Noordin for her

    suggestions, innovative ideas and also her assistance in collection of some samples

    used in this study. I genuinely appreciate her help. I am also thankful to Dr. Lim

    Sheh Hong for her time, guidance and also for sharing information and knowledge

    regarding this project.

    Moreover, I acknowledge the financial supports provided by Ministry of

    Higher Education (MOHE) for MyBrain15 and also by Universiti Sains Malaysia

    (USM) for the Geran Penyelidikan Siswazah Penyelidikan (RU-PRGS).

    Not forgetting the Industrial Biotechnology Research Laboratory (IBRL)

    members, primarily Dr. Tong Woei Yenn, and Miss Nor Afifah Supardy for their

    endless supports, care and valuable friendships.

    My special thanks goes to my father, mother, hubby and son for their

    undyeing love, continuous supports, advices, inspiration and motivations.

    Azlinah

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    TABLE OF CONTENTS

    Page

    ACKNOWLEDGEMENT ii

    TABLE OF CONTENTS iii

    LIST OF TABLES xvi

    LIST OF FIGURES xx

    LIST OF ABBREVIATIONS xxvi

    ABSTRAK xxvii

    ABSTRACT xxx

    CHAPTER 1.0 INTRODUCTION 1

    1.1 Problem statements 1

    1.2 Rational of study 2

    1.3 Research objectives 5

    1.4 Scope of study 5

    CHAPTER 2.0 LITERATURE REVIEW 7

    2.1 Definition of pigments and its classification 7

    2.2 Disadvantages of synthetic pigment 8

    2.2.1 Effect of synthetic pigments on environment 9

    2.2.2 Effect of synthetic pigment on human health 10

    2.3 Sources and distribution of natural pigments in nature 11

    2.3.1 Terrestrial environment (plants, animal, microbes) 12

    2.3.1.1 Terrestrial plants 12

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    2.3.1.2 Terrestrial animal 13

    2.3.1.3 Terrestrial microorganisms 14

    2.3.2 Marine environment 16

    2.3.2.1 Marine plants (Algae) 17

    2.3.2.2 Marine animal 17

    2.3.2.3 Marine microorganisms 18

    2.3.2.3.1 Marine bacteria 19

    2.3.2.3.1.1 Advantageous of microorganisms as

    pigment sources over plants and

    animals

    22

    2.3.2.3.1.2 Symbiosis 24

    2.3.2.3.1.3 Adaptation mechanism of bacteria to

    marine environment

    27

    2.3.2.3.1.4 Challenge in isolating marine bacteria 30

    2.3.2.3.1.5 Pseudoalteromonas sp. 31

    2.4 Functions of bacterial natural pigments 33

    2.4.1 Function of bacterial natural pigments to its origin (physiological

    roles) in marine environment

    33

    2.4.2 Function of bacterial natural pigments in industries 35

    2.4.2.1 Application of natural pigments in food, aquaculture,

    pharmaceutical and textile industries

    35

    2.5 Class of bacterial pigments 38

    2.6 Antibacterial pigments from marine bacteria 38

    2.6.1 Prodigiosin 41

    2.6.1.1 Structure and types of prodigiosin 41

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    2.6.1.2 Types of prodigiosin and its derivatives 42

    2.6.1.3 Physical and chemical properties of prodigiosin pigment 43

    2.6.1.4 Sources and distribution of prodigiosin 44

    2.6.1.5 Prospects and functions and applications of prodigiosin 44

    2.6.1.6 Application of prodigiosin in textile industry 45

    2.6.1.6.1 Textile industry 45

    2.6.1.6.2 Prospect of dyeing fabrics with natural pigments 46

    2.6.1.6.3 The need for antimicrobial finishing for fabrics 48

    2.6.1.6.4 Textile as microbial carrier 50

    2.6.1.6.5 Disadvantages of synthetic antimicrobial agent in

    textile fabrics

    51

    2.6.1.6.6 The use of natural antimicrobial agent for textile 52

    2.6.2.6.7 Application of natural prodigiosin pigment in

    textile industry

    53

    CHAPTER 3.0 SAMPLING AND ISOLATION OF MARINE

    BACTERIA

    54

    3.1 Introduction 54

    3.2 Materials and Methods 55

    3.2.1 Sample collection of marine resources 55

    3.2.2 Isolation 55

    3.2.2.1 Medium 55

    3.2.2.2 Antibiotics 56

    3.2.2.3 Isolation from seawater 56

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    3.2.2.4 Isolation from seaweeds, corals (soft and hard), snails,

    clamshell, stone, fish net, barnacles

    57

    3.2.2.5 Isolation from marine sediments 57

    3.2.2.6 Preservation of isolated bacteria 58

    3.2.3 Cultivability of isolated bacteria 58

    3.2.4 Characterization of isolated cultivable bacteria 59

    3.2.4.1 Morphological characterization (macroscopic study) 59

    3.2.4.2 Gram staining (microscopic study) 59

    3.2.4.3 Potassium hydroxide (KOH) test (Gregerson method) 60

    3.2.5 Observation of the surface of marine macroalgae with Scanning

    Electron Microscope (SEM)

    60

    3.3 Results and Discussion 61

    3.3.1 Sampling 61

    3.3.2 Isolation 64

    3.3.3 Cultivability of isolated marine bacteria 68

    3.3.4 Morphological characterization and Gram classification 71

    3.3.5 Observation of marine macro algae surface for marine bacteria

    colonization using Scanning Electron Microscope (SEM)

    75

    3.4 Conclusion 79

    CHAPTER 4.0 SCREENING FOR PIGMENT PRODUCTION AND

    ANTIMICROBIAL ACTIVITY

    80

    4.1 Introduction 80

    4.2 Materials and Methods 81

    4.2.1 Screening of isolates for pigment production 81

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    4.2.1.1 Media 81

    4.2.1.2 Seed culture preparation and cultivation 81

    4.2.1.3 Visual screening (Primary screening) 82

    4.2.1.4 Analytical screening (Secondary screening) 82

    4.2.1.4.1 Biomass determination 82

    4.2.1.4.2 Extraction 83

    4.2.1.4.3 UV/vis analysis 84

    4.2.2 Screening of isolates for antimicrobial activity using disc

    diffusion assay

    84

    4.2.2.1 Media 84

    4.2.2.2 Test microorganisms 86

    4.2.2.3 Preparation of crude extract 86

    4.2.2.4 Preparation of inoculum suspension 87

    4.2.2.4.1 Bacteria and yeasts 87

    4.2.2.4.2 Fungi 87

    4.2.2.5 Inoculation of test plate 88

    4.2.2.6 Application of disc onto inoculated agar 88

    4.2.2.7 Positive and negative controls. 89

    4.2.2.8 Reading plates and interpreting results 89

    4.2.3 Screening of isolates for antimicrobial activity using broth

    micro dilution assay

    90

    4.2.3.1 Media 90

    4.2.3.2 Preparation of crude extract 90

    4.2.3.3 Preparation of inoculum suspension 91

    4.2.3.4 Addition of extract and bacterial suspension 91

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    4.2.3.5 Determination of MIC value 92

    4.2.3.6 Minimum Bactericidal Concentration (MBC assay) 92

    4.2.4 Solvent effectiveness on extraction of intracellular pigment 93

    4.2.5 Screening for e