production of natural pigment with antimicrobial...
TRANSCRIPT
i
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
ii
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
iii
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
iv
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
v
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
vi
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
vii
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
viii
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 extracellular pigment for pigment production
and antimicrobial activity.
94
4.2.6 Solvent-solvent partitioning 94
4.2.6.1 MIC tests of partitioned extract 96
4.2.6.2 MBC tests of partitioned extract 98
4.3 Results and Discussion 99
4.3.1 Visual screening of colour production (primary screening) 99
4.3.2 Analytical screening of colour production (secondary
screening)
100
4.3.3 Antimicrobial activity determination using disc diffusion assay 108
4.3.4 Determination of Minimum Inhibitory Concentration (MIC)
and Minimum Bactericidal Concentration (MBC) of crude
extracts PD12 IBRL and BF1A IBRL
116
4.3.5 Screening of effective solvent in extracting intracellular
pigment
119
4.3.6 Extraction of extracellular pigments 126
4.3.7 Solvent-solvent partitioning process 129
4.3.7.1 Minimum Inhibitory Concentration (MIC) of isolate
BF1A IBRL before and after partitioning
137
4.4 Conclusion 139
ix
CHAPTER 5.0 CHARACTERIZATION AND IDENTIFICATION OF
THE POTENTIAL PIGMENT PRODUCING ISOLATE
141
5.1 Introduction 141
5.2 Materials and Methods 142
5.2.1 Macroscopic examination of isolate BF1A IBRL 142
5.2.2 Cultural characteristic study of isolate BF1A IBRL 142
5.2.3 Microscopic examination 143
5.2.3.1 Light microscopic study 143
5.2.3.2 Scanning electron microscopic (SEM) study 144
5.2.3.3 Transmission electron microscopic (TEM) study 145
5.2.3.3.1 Negative staining 145
5.2.3.3.2 Section of embedded materials 145
5.2.4 Biochemical test 147
5.2.4.1 Methyl red test 149
5.2.4.2 Catalase test 149
5.2.4.3 Oxidase test 149
5.2.4.4 Hydrogen sulfide test 150
5.2.4.5 Citrate utilization test 150
5.2.4.6 Characterization by API 20NE 151
5.2.5 Molecular characterization (Sequence comparison and
phylogenetic analysis)
152
5.3 Results and Discussion 152
5.3.1 Morphological characteristics of isolate BF1A IBRL
(Macroscopic examination)
152
5.3.2 Cultural characteristics of isolate BF1A IBRL 155
x
5.3.3 Microscopic examination of isolate BF1A IBRL 158
5.3.3.1 Light microscope and Gram staining 158
5.3.3.2 Scanning Electron Microscope 159
5.3.3.3 Transmission Electron Microscope 159
5.3.3.3.1 Negative staining 159
5.3.3.3.2 Section of embedded materials 162
5.3.4 Physical and biochemical characterization 164
5.3.5 Molecular characterization based on 16S r RNA gene 167
5.4 Conclusion 173
CHAPTER 6.0 PIGMENT PRODUCTION AND OPTIMIZATION
PROCESS OF CULTURAL CONDITION OF Pseudoalteromonas rubra
BF1A IBRL
174
6.1 Introduction 174
6.2 Materials and Methods 175
6.2.1 Characterizations of pigment produced by potential isolate, P.
rubra BF1A IBRL using UV/vis spectroscopic analysis
175
6.2.2 Standard curve of prodigiosin 175
6.2.3 Growth, pigment production and antibacterial
activity
176
6.2.3.1 Assay for antibacterial activity 176
6.2.4 Physical optimization process 177
6.2.4.1 Light and darkness condition 178
6.2.4.2 pH 178
6.2.4.3 Temperature 178
xi
6.2.4.4 Agitation speed 179
6.2.4.5 Inoculum size 179
6.2.5 Quantification of prodigiosin 179
6.2.6 Time Kill Study 180
6.2.7 Physical characterization of prodigiosin pigment in diethyl
ether fraction of P. rubra BF1A IBRL
181
6.2.7.1 Effect of temperature towards stability of prodigiosin
pigment
181
6.2.7.2 Effect of pH towards stability of prodigiosin pigment 182
6.2.7.3 Effect of Light towards stability of prodigiosin
pigment
182
6.2.7.4 Effect of incubation time towards stability of
prodigiosin pigment
183
6.2.8 Statistical analysis 184
6.3 Results and Discussions 184
6.3.1 Characterization of pigment produced by P. rubra BF1A
IBRL
184
6.3.2 Growth profile of isolate BF1A IBRL in relation to its
pigment production and antibacterial property
188
6.3.3 Optimization 192
6.3.3.1 Effect of light 192
6.3.3.2 pH optimization 195
6.3.3.3 Temperature optimization 197
6.3.3.4 Agitation speed optimization 201
xii
6.3.3.5 Inoculum size optimization 205
6.3.4 Quantification of prodigiosin produced by P. rubra BF1A
IBRL in batch production
208
6.3.5 Time Killing assay of MRSA in the presence of prodigiosin
extract
212
6.3.6 Stability of pigments 215
6.4 Conclusion
222
CHAPTER 7.0 CHEMICAL PROFILING AND BIOASSAY GUIDED
SEPARATION OF PIGMENT EXTRACT FROM Pseudoalteromonas
rubra BF1A IBRL
223
7.1 Introduction 223
7.2 Materials and Methods 224
7.2.1 Thin Layer Chromatography (TLC) 224
7.2.2 Antibacterial activity test of diethyl ether partition extract on
TLC chromatogram
225
7.2.2.1 Bioautography assay using agar overlay method 226
7.2.3 Column Chromatography (CC) technique 226
7.2.3.1 Column packing and development 226
7.2.3.2 Spectrophotometric analysis of fractions 227
7.2.3.3 Antimicrobial activity test of fraction 228
7.2.3.3.1 Sensitivity test of fractions 228
7.2.3.3.2 MIC tests of fraction 228
7.2.3.3.3 MBC tests of fraction 230
xiii
7.2.3.3.4 Structural degeneration of the fraction-
treated MRSA cells
230
7.2.4 Preparative TLC for purification 231
7.2.4.1 Spectrophotometric analysis of preparative TLC
purified compound
232
7.2.4.2 MIC and MBC determination of preparative TLC
purified compound
232
7.2.5 High Performance Liquid Chromatography (HPLC) 233
7.2.6 In vivo toxicity study 234
7.2.6.1 Preparation of artificial seawater (ASW) and
hatching of brine shrimp (Artemia salina)
234
7.2.6.2 Preparation of pigment extract 235
7.2.6.3 Brine shrimp lethality test (BLST) 237
7.3 Results and Discussion 238
7.3.1 Thin Layer Chromatography (TLC) 238
7.3.2 Bioautography detection in TLC of diethyl ether extract 242
7.3.3 Purification by Column Chromatography 245
7.3.3.1 Thin Layer Chromatographic analysis of fractions 245
7.3.3.2 Spectroscopic analysis of fractions 248
7.3.3.3 Bioassay guided fractionation compounds from P.
rubra BF1A IBRL
249
7.3.3.3.1 Sensitivity test of fractions 249
7.3.3.3.2 Minimum inhibitory concentration
(MIC) of active fraction
251
xiv
7.3.3.3.3 Morphological changes of MRSA treated
with active fraction (fraction 4) of P. rubra
BF1A IBRL
254
7.3.4 Preparative TLC 259
7.3.5 High performance of Liquid Chromatography (HPLC) 261
7.3.6 In vivo toxicity of extract P. rubra BF1A IBRL 266
7.4 Conclusion 273
CHAPTER 8.0: APPLICATION OF PRODIGIOSIN PIGMENT FROM
Pseudoalteromonas rubra BF1A IBRL AS COLOURING AGENT AND
ANTIMICROBIAL AGENT IN TEXTILE INDUSTRY
274
8.1 Introduction 274
8.2 Materials and Methods 275
8.2.1 Fabrics materials 275
8.2.2 Mordants 275
8.2.3 Perspiration solutions 276
8.2.4 Pre-treatment of fabrics 276
8.2.5 Dye materials 276
8.2.6 Dyeing method using natural prodigiosin pigment 277
8.2.7 Dyeing method using reactive dye (synthetic pigment) 277
8.2.8 Mordanting procedure 277
8.2.9 Colourfastness standard test 278
8.2.9.1 Colourfastness to light 278
8.2.9.2 Colourfastness to washing 279
8.2.9.3 Colourfastness to rubbing/ crocking 279
xv
8.2.9.4 Colour fastness to perspiration 280
8.2.9.5 Colourfastness to water 281
8.2.10 Antibacterial evaluation test of prodigiosin-dyed fabric 281
8.3 Results and Discussion 282
8.3.1 Dyeability of prodigiosin on different fabrics 282
8.3.2 Dyeability of prodigiosin on different fabrics using
mordants
284
8.3.3 Comparison of fastness properties of prodigiosin with
Reactive dyes
293
8.3.4 Antibacterial evaluation test of prodigiosin-dyed fabric 296
8.4 Conclusion 302
CHAPTER 9.0 GENERAL CONCLUSION AND RECOMENDATIONS
FOR FUTURE STUDY
304
REFERENCES 307
APPENDIX 347
xvi
LIST OF TABLES
Page
Table 2.1 Examples of pigment producing terrestrial plants
14
Table 2.2 Marine bacteria that were able to produce various pigmented
bioactive compounds
21
Table 2.3 Bacteria that have been isolated from surface of higher
marine organisms producing various types of non-pigmented
compounds
26
Table 2.4 Bacteria that have been isolated from surface of higher
marine organisms producing various types of pigmented
compounds
27
Table 2.5 Examples and colour characteristics of marine bacterial
pigments of biological importance
39
Table 2.6 Some antibiotic pigments isolated from marine bacteria
41
Table 2.7 Some natural pigments used in textile dyeing
47
Table 2.8 Disadvantages of some synthetic antimicrobial agent used in
textile industry
52
Table 3.1 Sampling location and condition
62
Table 3.2 List of isolated marine bacteria according to sampling
location
66
Table 3.3 Cultivability of isolated bacteria in relation to its isolation
source.
69
Table 3.4 Macroscopic characterization and Gram classification of
isolated bacteria
72
Table 4.1: Preparation of final inoculum concentration from stock
suspension
91
Table 4.2: Scheme for preparing dilution series of water insoluble
partitioned extract to be used in MIC assay
99
Table 4.3: UV/vis characterization of pigments produced by marine
bacteria
103
Table 4.4: Anti Antimicrobial activity of the isolated marine bacteria by disc
diffu diffusion assay
109
xvii
Table 4.5: Summary of antimicrobial spectrum of isolated marine
bacteria in regards to its isolation source
113
Table 4.6: The MIC and MBC values of intracellular crude extract of
PD12 IBRL and BF1A IBRL on test bacteria
118
Table 4.7: Property of intracellular extract of isolate BF1A IBRL using
different extractants
120
Table 4.8: Property of intracellular extract of isolate PD12 IBRL using
different extractants
122
Table 4.9 Property of intracellular extract of isolate BF11 IBRL using
different extractants
123
Table 4.10: Property of extracellular extract of isolate BF1A IBRL,
PD12 IBRL, and BF11 IBRL
127
Table 4.11: Summary of antimicrobial activity of intracellular and
extracellular extract of isolate BF1A IBRL, PD12 IBRL, and
BF11 IBRL
128
Table 4.12: Colour property of extract BF1A IBRL, PD12 IBRL and
BF11 IBRL from solvent partitioning
131
Table 4.13: Total yield of extract BF1A IBRL, PD12 IBRL and BF11
IBRL from solvent partitioning
132
Table 4.14: Absorption spectrum of pigments of BF1A IBRL, PD12
IBRL and BF11 IBRL in different partitions
133
Table 4.15: Antimicrobial activity of different partitions extract of
isolate BF1A IBRL, PD12 IBRL, BF11 IBRL
134
Table 4.16: MIC values of partitioned extract compared to ethyl acetate
extract (crude) and positive control
138
Table 4.17: MBC values of partitioned extract compared to ethyl acetate
extract (crude) and positive control
139
Table 5.1: Different types of medium compositions used for growth and
pigment production
143
Table 5.2 : Dehydration process in cell preparation for SEM observation
144
Table 5.3 : Dehydration process in cell preparation for TEM observation
146
Table 5.4 : Morphological characteristics of a marine isolate BF1A
IBRL
155
xviii
Table 5.5: Ability of isolate BF1A IBRL to grow and produce pigment
in different types of medium
156
Table 5.6 : Physical and biochemical characteristics of the isolate BF1A
IBRL
165
Table 6.1: Quantification of extracellular pigment from P. rubra BF1A
IBRL
210
Table 7.1: Scheme for preparing dilution series of water insoluble
extract (Fraction 4) to be used in MIC assay
229
Table 7.2: Scheme for preparing dilution series of water insoluble
extract (preparative-TLC purified extract) to be used in MIC
assay
233
Table 7.3: Scheme for preparing dilution series of water insoluble ethyl
acetate extract (crude) to be used in brine shrimp toxicity test
236
Table 7.4: Scheme for preparing dilution series of water insoluble
diethyl ether partition extract to be used in brine shrimp
toxicity test
236
Table 7.5: Scheme for preparing dilution series of fraction 4 to be used
in brine shrimp toxicity test
237
Table 7.6: TLC: Retention factor (Rf) values and colour properties of
the detected spots of diethyl ether partition extract of P.
rubra BF1A IBRL
240
Table 7.7: Inhibitory spots detected from bioautography assay using
agar overlay method
243
Table 7.8 The TLC analysis of the fractions collected from column
chromatography.
246
Table 7.9: Sensitivity test results of fractionated extracts (2 mg/disc) of
P. rubra BF1A IBRL against MRSA
250
Table 7.10: The MIC and MBC values of Fraction 4 in comparison with
MIC and MBC values of crude and partitioned diethyl ether
extract of P. rubra BF1A IBRL
252
Table 7.11: The yield of prodigiosin in different samples of P. rubra
BF1A IBRL
265
Table 7.12: Summary of toxicity levels of extracts obtained from P.
rubra BF1A IBRL
272
xix
Table 8.1: Shades and fastness properties of different fabrics dyed with
prodigiosin extract and mordanted using lime
288
Table 8.2: Shades and fastness properties of different fabrics dyed with
prodigiosin extract and mordanted using copper sulphate
289
Table 8.3: Shades and fastness properties of wool fabrics dyed with
prodigiosin extract and mordanted using different mordant
292
Table 8.4: Comparison of shades and fastness properties of wool
fabrics dyed with prodigiosin extract and synthetic dye
294
Table 8.5: Antibacterial evaluation of wool fabrics dyed with bacterial
prodigiosin pigment extract against different bacteria
297
xx
LIST OF FIGURES
Page
Figure 2.1 Structures of some prodigionine
43
Figure 3.1 Some of the animate marine resources collected from
different sampling sites
63
Figure 3.2 Classification of isolated marine bacteria based on colour
property and also Gram reaction
75
Figure 3.3 Epibiotic colonization on the surfaces of macroalgal
(Caulerpa sp) observed by scanning electron microscope
(SEM)
76
Figure 3.4 Epibiotic colonization on the surface of macroalgal
(Sargasum sp) observed by scanning electron microscope
(SEM)
77
Figure 4.1 Flowchart of pigment extraction of intracellular and
extracellular extract of marine bacteria
85
Figure 4.2 Flow Chart of organic solvent extraction and solvent-
solvent partitioning
97
Figure 4.3 Example of pigmentation produced by some of the marine
bacteria.
100
Figure 4.4 Dry cell weight of crude extract obtained from pellet
(intracellular) and supernatant (extracellular) of the isolates
101
Figure 4.5 Absorption spectrum pattern of pigment extracts that similar
to the absorption spectrum of carotenoids pigment
106
Figure 4.6 Absorption spectrum pattern of pigment extracts that likely
to be pigments other than carotenoids
107
Figure 4.7 Crude extract from biomass BF1A IBRL extracted with
different solvents
120
Figure 4.8 Crude extract from biomass PD12 IBRL extracted with
different solvents
121
Figure 4.9 Crude extract from biomass BF11 IBRL extracted with
different solvents
123
Figure 4.10 Disc diffusion assay result of diethyl ether partition of
supernatant BF1A IBRL
136
xxi
Figure 5.1 Diagrammatic scheme for the identification of Gram
negative marine bacteria
148
Figure 5.2 Colony morphology of isolate BF1A IBRL grown on
Marine agar 2216 plates
153
Figure 5.3 A closer look of 3 days old colony BF1A IBRL on Marine
Agar
154
Figure 5.4 The colonies of isolate BF1A IBRL as observed under a
dissecting microscope
154
Figure 5.5 Shake flasks containing Medium 1 (marine broth 2216)
before and after inoculation of isolate BF1A IBRL
157
Figure 5.6 Light micrograph of isolate BF1A IBRL
158
Figure 5.7 SEM micrograph of BF1A IBRL grown in Medium 1 (MB
2216) for 24 hours at 26°C and 120 rpm of agitation
160
Figure 5.8 TEM micrographs of BF1A IBRL show the monotrichus
flagellum on the cell
161
Figure 5.9 TEM micrographs of isolate BF1A IBRL grown for 24
hours
163
Figure 5.10 Some biochemical test results obtained from conventional
and commercial kit identification methods
166
Figure 5.11 The PCR amplicon of 16S rRNA gene obtained from isolate
BF1A IBRL
167
Figure 5.12 The consensus sequences of isolate BF1A IBRL
168
Figure 5.13 Top 10 of BLASTn Algorthm search’s results which
matched with isolate BF1A IBRL
169
Figure 5.14 Phylogenetic tree based on bacterial 16S rRNA gene
sequence of the isolate BF1A IBRL strain and other species
of genus Pseudoalteromonas.
170
Figure 6.1 UV/vis spectrum of pigment in diethyl ether extract of P.
rubra BF1A IBRL and standard prodigiosin
185
Figure 6.2 UV/vis spectrum of pigment in diethyl ether extract of P.
rubra BF1A under acidic and alkaline condition
185
Figure 6.3 Presumptive test for prodigiosin
186
xxii
Figure 6.4 Standard prodigiosin calibration curve
188
Figure 6.5 Time course profile of growth, pigment production and anti-
MRSA activity of diethyl ether extract of P. rubra BF1A
IBRL
190
Figure 6.6 Pigment extract paste (redissolved in ethanol) obtained at
different cultivation period of P. rubra BF1A IBRL
191
Figure 6.7 Effect of light on prodigiosin production, antibacterial
activity and growth of P. rubra BF1A IBRL
193
Figure 6.8 Marine broth after cultivated with P. rubra BF1A IBRL for
24 hours at 26 °C, and 120 rpm at dark condition and light
conditions
194
Figure 6.9 Pigment extract paste (re-dissolved in ethanol) obtained
after cultivation of P. rubra BF1A IBRL at dark and light
condition
194
Figure 6.10 Effect of various pH on prodigiosin production,
antibacterial activity and growth of P. rubra BF1A IBRL
196
Figure 6.11 Marine broth with different pH after cultivated with P.
rubra BF1A IBRL for 24 hours at 26 °C, and 120 rpm
197
Figure 6.12 Pigment extract paste (re-dissolved in ethanol) obtained
after cultivation of P. rubra BF1A IBRL in different pH of
MB
197
Figure 6.13 Effect of various temperatures on prodigiosin production,
antibacterial activity and growth of P. rubra BF1A IBRL
198
Figure 6.14 Marine broth after cultivated with P. rubra BF1A IBRL for
24 hours at 120 rpm and at different incubation temperature
200
Figure 6.15 Pigment extract paste (re-dissolved in ethanol) obtained
after cultivation of P. rubra BF1A IBRL in different
incubation temperature
200
Figure 6.16 Effect of various agitation speed on prodigiosin production,
antibacterial activity and growth of P. rubra BF1A IBRL
202
Figure 6.17 Marine broth after cultivated with P. rubra BF1A IBRL for
24 hours at 26 °C, and agitated at different agitation speed
202
Figure 6.18 Pigment extract paste (re-dissolved in ethanol) obtained
after cultivation of P. rubra BF1A IBRL at different
agitation speeds
203
xxiii
Figure 6.19 Cell structure and arrangement of P. rubra BF1A IBRL as
observed with Light microscope (100X) at different
agitation speeds on 24 hours of fermentation
204
Figure 6.20 Effect of various inoculums size on prodigiosin production,
antibacterial activity and growth of P. rubra BF1A IBRL
206
Figure 6.21 Marine broth after cultivated with P. rubra BF1A IBRL for
24 hours at 26 °C, and 120 rpm using different inoculums
sizes.
207
Figure 6.22 Pigment extract paste (re-dissolved in ethanol) obtained
after cultivation of P. rubra BF1A IBRL of various
inoculum size
207
Figure 6.23 Marine broth cultivated with P. rubra BF1A IBRL in 1000
mL shake flask
209
Figure 6.24 Marine broth cultivated with P. rubra BF1A IBRL in 250
mL shake flask
209
Figure 6.25 Pigment extract of P. rubra BF1A IBRL after redissolving
in ethanol at concentration of 100 mg/mL
211
Figure 6.26 Time kill study of MRSA exposed to diethyl ether extract of
spent broth P. rubra BF1A IBRL at different concentration
varied from 27.5 to 55.0 µg/mL
213
Figure 6.27 Stability of prodigiosin extract at different temperature
216
Figure 6.28 Tubes containing pigment in aqueous form which was
treated with different high temperatures
217
Figure 6.29 Stability of prodigiosin at different pH
218
Photo 6.30 Petri dish of disc diffusion assay shows the zone of
inhibition by the diethyl ether partition extract after treated
with different pH for 30 minutes
218
Figure 6.31 Stability of prodigiosin towards illumination
220
Figure 6.32 Petri dish of disc diffusion assay shows the zone of
inhibition by the DE partition extract incubated at light and
dark condition
221
Figure 6.33 Effect of incubation time on stability of the prodigiosin
extract
222
Figure 7.1 The intermediate concentrations of Fraction 4
230
xxiv
Figure 7.2 Chromatograms of diethyl ether partition extract of BF1A
IBRL developed using Acetone: hexane (5:5)
241
Figure 7.3 Graphical elaboration: chromatogram of diethyl ether
partition extract of BF1A IBRL developed using Acetone:
hexane (5:5)
241
Figure 7.4 Antibacterial activity of TLC chromatograms of diethyl
ether partition extract of P. rubra BF1A IBRL
244
Figure 7.5 Chromatograms developed using acetone: hexane (5:5) of
standard prodigiosin and diethyl ether extract (before and
after fractionation)
247
Figure 7.6 The absorbance of different fractions collected from column
chromatography of diethyl extract of P. rubra BF1A IBRL
248
Figure 7.7 Inhibition zone of different fractions against MRSA
250
Figure 7.8 SEM micrograph showing the effect of Fraction 4 on
MRSA at different time of exposure
255
Figure 7.9 TEM micrograph showing the effect of Fraction 4 on
MRSA at different time of exposure
258
Figure 7.10 Characteristic UV- visible of standard prodigiosin and
preparative TLC purified compound from P. rubra BF1A
IBRL bacterium.
259
Figure 7.11 HPLC chromatogram of diethyl ether partition extract of P.
rubra BF1A IBRL (Before column chromatography)
262
Figure 7.12 HPLC chromatogram of Fraction 4 of P. rubra BF1A IBRL
(After column chromatography)
262
Figure 7.13 HPLC chromatogram of TLC-prep purified compounds of
P. rubra BF1A IBRL (After column chromatography and
TLC-preparative)
263
Figure 7.14 HPLC chromatogram of standard prodigiosin
264
Figure 7.15 HPLC chromatogram of the standard prodigiosin and TLC-
prep purified compound of P. rubra BF1A IBRL
264
Figure 7.16 Toxicity result of extracellular ethyl acetate extract (crude)
of Pseudoalteromonas rubra BF1A IBRL against brine
shrimp after 6 hours of exposure time (for acute cytotoxicity
test)
267
xxv
Figure 7.17 Toxicity result of extracellular ethyl acetate extract (crude)
of Pseudoalteromonas rubra BF1A IBRL against brine
shrimp after 24 hours of exposure time (for chronic
cytotoxicity test)
268
Figure 7.18 Toxicity result of diethyl ether partition extract of P. rubra
BF1A IBRL against brine shrimp after 6 hours of exposure
time (for acute cytotoxicity test)
269
Figure 7.19 Toxicity result of diethyl ether partition extract of P. rubra
BF1A IBRL supernatant extract against brine shrimp after
24 hours of exposure time (for chronic cytotoxicity test)
269
Figure 7.20 Toxicity result of Fraction 4 of P. rubra BF1A IBRL
against brine shrimp after 6 hours of exposure time (for
acute cytotoxicity test)
270
Figure 7.21 Toxicity result of Fraction 4 of P. rubra BF1A IBRL
against brine shrimp after 24 hours of exposure time (for
chronic cytotoxic test)
270
Figure 8.1 The blue wool standard used to evaluate the colour changes
due to exposure to light
279
Figure 8.2 Dyestuff of natural prodigiosin extract, synthetic 3BS and
synthetic Red Chilli dye
283
Figure 8.3 Dyeability of prodigiosin extract on different fabrics
without using mordant
284
Figure 8.4 Dyeability of prodigiosin extract on different fabrics using
slaked lime as mordant
285
Figure 8.5 Dyeability of prodigiosin extract on different fabrics using
copper sulphate as mordant
286
Figure 8.6 Colour of dyed wool fabric with and without mordant
291
Figure 8.7 Comparison of dyeing performance of wool fabric using
synthetic dye and natural dye
293
Figure 8.8 The pictures of antibacterial activity of control wool fabric
and dyed wool fabric using natural prodigiosin extract
against B. subtilis, B. cereus and MRSA, and S.aureus
300
Figure 8.9 The pictures of antibacterial activity of control wool fabric
and dyed wool fabric using natural prodigiosin extract
against A. anitratus, S. epidermidis, K. pneumoniae, and E.
coli.
301
xxvi
LIST OF ABBREVIATIONS
ACE Acetone
ATCC American Type Culture Collection
CCB Centre for Chemical Biology
CFU Colony Forming Unit
CHL Chloroform
CV-I Crystal violet-Iodine
DE Diethyl ether
EtOAc Ethyl acetate
FDA Food and Drug Administration
H2O2 Sulphuric acid
HCL Hydrochloric acid
HMDS Hexamethyldisilazine
HPLC High Performance Liquid Chromatography
Hx Hexane
INT p-iodonitrotetrazolium violet
LC50 50% lethal concentration
MA Marine Agar
MAP 2-methyl-3-n-amyl-pyrrole
MBC Minimum Bactericidal Concentration
MBC 4-methoxy-2,2’-bipyrrole-5-carbaldehyde
MHA Mueller Hinton Agar
MHB Mueller Hinton Broth
MIC Minimum Inhibitory Concentration
MM Minimal medium
MRSA Methicillin-resistance Staphylococcus aureus
NA Nutrient Agar
NaOH Sodium hydroxide
NCBI National Centre for Biotechnology Information
OD Optical density
P Iso-propanol
P.I Polarity index
PDA Potato Dextrose Agar
r/t Retention time
Rf Retention factor
SDA Sabouraud Dextrose Agar
SDC The Society of Dyers and Colourists
SEM Scanning Electron Microscope
TEM Transmission electron microscope
TLC Thin Layer Chromatography
UV/vis Ultra-violet visible
xxvii
PENGHASILAN PIGMEN SEMULA JADI DENGAN AKTIVITI
ANTIMIKROB DARIPADA BAKTERIA MARIN,
Pseudoalteromonas rubra BF1A IBRL
ABSTRAK
Pigmen semula jadi adalah alternatif kepada pewarna sintetik yang sering
digunakan dalam industri tekstil, kosmetik, makanan, farmaseutikal dan juga industri
akuakultur. Bakteria marin adalah salah satu sumber biologi yang produktif dalam
menghasilkan pigmen semula jadi. Selain berfungsi untuk mewarna, pigmen semula
jadi juga telah dilaporkan mempunyai fungsi antimikrob. Kajian untuk penemuan
sebatian antimikrob masih berterusan dan dengan itu, kajian mengenai sebatian
antimikrob berwarna turut dapat membantu mengembangkan lagi penyelidikan
antimikrob tersebut di mana sebatian antimikrob yang berwarna dapat memberikan
lebih manfaat kepada pelbagai industri. Hal ini dapat berlaku kerana sebatian
tersebut bukan sahaja berfungsi untuk mewarna malah turut dapat menjadi bahan
pengawet pada masa yang sama bagi sesuatu produk. Justeru itu, kajian ini
dijalankan untuk menghasilkan pigmen semula jadi daripada bakteria marin yang
dikulturkan di dalam sistem kelalang dan juga mengkaji aktiviti antibakteria yang
dipamerkan oleh pigmen tersebut. Dalam kajian ini, sebanyak 30 bakteria berpigmen
telah dipencilkan daripada sampel marin, termasuk 8 daripada makroalga, 8 daripada
batu karang, 6 daripada sumber haiwan laut, 6 daripada pasir laut, 1 daripada air laut
dan 1 daripada sangkar ikan. Kebanyakan bakteria yang dipencilkan adalah
berpigmen kuning (53%), diikuti dengan jingga (30%), merah jambu (10%) dan
merah (7%). Keputusan analisis gravimetrik bagi penghasilan pigmen menunjukkan
bahawa semua pencilan dapat menghasilkan pelbagai jenis pigmen pada kuantiti
xxviii
yang berbeza, iaitu dalam julat 0.294 g / L hingga 1.542 g / L. Daripada 30 pencilan
bakteria, didapati sebanyak 9 bakteria mempamerkan aktiviti antibakteria terhadap
sekurang-kurangnya satu bakteria ujian. Daripada 9 jenis bakteria ini pula, 1 bakteria
dipilih iaitu bakteria BF1A IBRL sebagai bakteria berpotensi kerana ia dapat
merencat lebih banyak bakteria ujian dengan nilai MIC yang lebih rendah, iaitu di
antara 0.28 -8.88 mg /mL. Pigmen ekstrasel yang dihasilkan oleh pencilan BF1A
IBRL mempamerkan aktiviti antibakteria yang lebih baik berbanding dengan pigmen
intrasel. Pencilan BF1A IBRL telah dikenalpasti sebagai Pseudoalteromonas rubra
berdasarkan ciri-ciri fenotip dan genotip. Keputusan yang diperolehi daripada analisa
UV/vis spektoskopi, ujian pigmen dan analisa kromatografi menunjukkan bahawa
pigmen yang dihasilkan oleh P. rubra BF1A IBRL adalah jenis prodigiosin iaitu
pigmen ini berwarna merah dan menunjukkan penyerapan serapan maksimum
UV/vis pada 534 nm. Dalam kajian masa maut, aktiviti antibakteria ekstrak
prodigiosin P. rubra BF1A IBRL adalah bergantung kepada kepekatan ekstrak.
Keputusan daripada proses penyisihan dan penulenan mendedahkan bahawa pigmen
prodigiosin yang tulen mempamerkan aktiviti antimikrob yang lebih baik berbanding
dengan sebatian campuran. Fraksi aktif (Fraksi 4) yang diperolehi daripada
kromatografi turus mempunyai nilai kepekatan perencatan minimum yang sama
dengan pigmen prodigiosin yang ditulenkan menggunakan preparative-TLC, iaitu
13.75 μg / mL terhadap B. subtilis, B. cereus, MRSA, S. aureus, dan A. anitratus.
Prodigiosin yang tulen adalah sangat toksik kepada Artemia salina bagi kedua-dua
tahap akut dan kronik. Namun ekstrak prodigiosin tidak menunjukkan sifat toksik
terhadap anak udang tersebut. Keupayaan pigmen prodigiosin dalam mewarnakan
fabrik tekstil telah dikaji dan didapati prodigiosin mampu mewarnakan fabrik wol
dengan lebih baik berbanding dengan fabrik kapas, satin sutera dan sutera. Kain wol
xxix
yang telah dicelup dengan pewarna semula jadi menunjukkan daya ketahanan yang
baik terhadap kesemua ujian ketahanan kecuali cahaya. Tambahan pula, fabrik wol
yang dicelup dengan prodigiosin semula jadi menunjukkan 97.21% kepada 99.99%
perencatan bakteria apabila fabrik yang ditambah dengan B. subtilis, B. cereus,
MRSA, S. aureus, A. anitratus dan S. epidermidis, manakala 2.83% kepada 49.8%
perencatan bakteria telah dicapai untuk E. coli dan K. pneumoniae. Secara
keseluruhan, pigmen semula jadi yang diekstrak daripada Pseudoalteromonas rubra
BF1A IBRL mempunyai potensi untuk menjadi bahan pewarna dan agen antibakteria
sekaligus.
xxx
PRODUCTION OF NATURAL PIGMENT WITH ANTIMICROBIAL
ACTIVITY FROM A MARINE BACTERIUM,
Pseudoalteromonas rubra BF1A IBRL
ABSTRACT
Natural pigments are important substitutes to the synthetic dyes in many
industrial applications including textile, cosmetics, food, pharmaceutical and also
aquaculture industry. Marine bacteria is one of the prolific biological sources for the
natural pigments. Besides colouring benefits, many natural pigments posses
antimicrobial properties. The search of antimicrobial agents is a continuing dialogue
to fight different types of diseases. Thus, the finding of colorant antimicrobial agent
can expand the current portrait of the research and can give benefits to many
industries since the colorants may give both tinctorial and preservative value
simultaneously to the industrial products. The aim of the study is to produce natural
pigment by cultivating the marine bacteria in shake flask system, and evaluate the
antimicrobial activity of the natural pigment. In this study, a total of 30 cultivable
pigmented bacteria were isolated from marine samples, including 8 from macroalgae,
8 from corals, 6 from animal resources, 6 from sand sediment, 1 from seawater and 1
from fish net. Most of the isolated strains were yellow pigmented bacteria (53%),
followed by orange (30%), pink (10%) and red pigmented bacteria (7%). Gravimetric
analysis of pigment production revealed that all the isolated strains were able to
produce different types of coloration with different quantities, where the crude
pigment yield ranged from 0.294 g/L to 1.542 g/L. Out of the 30 isolated bacteria, 9
bacteria exhibited antibacterial property against at least one test bacteria. Out of the 9
xxxi
selected strains, 1 isolate, namely BF1A IBRL was selected as potential strain since
it was able to inhibit broader range of test bacteria with lower MIC value, which
ranged from 0.28 to 8.88 mg/mL. The extracellular pigment of isolate BF1A IBRL
showed higher pigmentation strength with greater antimicrobial activity compared to
intracellular pigment. Isolate BF1A IBRL was identified as Pseudoalteromonas
rubra based on its phenotypic and genotypic characteristics. The results obtained
from UV/vis spectrophotometer, presumptive test and chromatographic analysis
indicated that the pigment produced by P. rubra BF1A IBRL is prodigiosin-type.
The pigment was red and showed a maximum absorption at 534 nm. Based on the
time kill assay the antibacterial activity of prodigiosin extract from P. rubra BF1A
IBRL was concentration dependant. The results of bio-guided purification analysis
revealed that the purified prodigiosin pigment exhibited greater antimicrobial activity
compared to the crude extract. The active fraction (Fraction 4) obtained from column
chromatography had similar MIC values with TLC-purified prodigiosin which was
13.75 µg/mL against B. subtilis, B. cereus, MRSA, S. aureus, and A. anitratus. The
purified prodigiosin was highly toxic towards the Artemia salina for both acute and
chronic toxicities; however the crude extract of the prodigiosin did not cause any
toxic effect towards the brine shrimp. The production and evaluation of microbial
pigment as textile colorants was investigated. The wool fabrics showed greater
affinity towards the prodigiosin extract compared to cotton, silk satin and silk. The
naturally dyed wool fabric showed fair to excellent fastness property towards all
colourfastness properties except for light colourfastness. Furthermore, the results also
disclose that the wool dyed with natural prodigiosin showed 97.21% to 99.99% of
bacterial reduction when the fabric was treated with B. subtilis, B. cereus, MRSA, S.
aureus A. anitratus and S. epidermidis, whereas 2.83% to 49.8% of bacterial
xxxii
reduction was achieved for E. coli and K. pneumoniae. Overall, the pigment
component isolated from Pseudoalteromonas rubra BF1A IBRL has potential as
colorant and preservative agent.
1
CHAPTER 1.0 INTRODUCTION
1.1 Problem statement
Synthetic pigments have been widely used as colouring agent in many
industries including food, textile, cosmetics and also aquaculture. The synthetic
pigments are created through chemical manufacturing, and exhibit toxic,
carcinogenic and mutagenic properties, thus create lots of adverse effect on human
and environments (Pathak & Chauhan, 2013). Recently, consumers awareness on
natural products are alarming as a consequence of proven toxicological effect of
some synthetic compounds, limited chemical diversity and structural complexity, and
also the great success of natural products on the market in the last years. This
consumer awareness has necessitated the need to explore for natural pigments from
natural sources as an alternative to the synthetic pigments.
It is well known that the number of drug resistance pathogens have increased
over time. The survival of these antibiotic-resistance pathogens in hospitals
environments, hospitals fabrics and hospital worker’s uniforms are the growing
concern particularly in units in which patients are immunosuppressed either
intentionally (as for transplantation) or as a result of trauma (severe burns) or disease
(such as acquired immunodeficiency disease). Fabrics are known to be the vector for
spreading the harmful bacteria (Neely & Maley, 2000). The most common harmful
bacteria are Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas
aeruginosa, Staphylococcus epidermidis, Klebsiella pneumoniae, and Enterobacter
aerogenes (Lee et al., 2009). As more bacteria have the ability to survive on fabrics,
and some even become resistant to antibiotics, hence the ability to control the spread
of these bacteria with antibiotic treatments decreases. Therefore, the research and
2
investigation to develop fabrics with eco-friendly antibacterial finishing is vital in
recent years.
1.2 Rational of study
Pigments are colourful compounds consisting of chromaphores, which either
absorb or reflect light in varying wavelengths of the visible region (Delgado-Vargas
et al., 2000). It is known that colour is the crucial factor of quality in some industrial
product to be commercialized and also an important attribute that determine the
consumer’s acceptance. Hence, pigments are used as colouring agent for colouring
the paints, plastics, inks, fabrics, cosmetics, food and other materials.
Natural pigments are safer, healthier, biodegradable, and exhibit higher
compatibility with the environment. Hence, the screening process for biological
source for new pigment is expected to be continued in the future. The natural sources
of colorants have emerged at recent times, where the natural pigments can be either
produced by plants (higher and lower plants) and also microorganisms.
Among the natural sources of pigments, microorganisms offer great
advantages for the pigment production of commercial activities. Microorganisms are
ubiquitous and an indispensable component of our industries. Moreover, microbial
pigments provide a safer alternative to chemical synthetic dyes and an economical
substitute for natural pigments obtained from plants and animals. Although
microorganisms are distributed in both terrestrial and marine environments, and most
of the bioactive compounds have been isolated from both environments, but still the
marine microorganism are more attractive to researchers. This is because the marine
microorganisms, particularly marine bacteria have potential to produce compounds
3
with unique biological properties, this serves as an attractive choice for commercial
scale production. Moreover, one of the main goals of the marine biotechnology
recently is the finding of natural substances originated from marine microorganisms
(Darabpour et al., 2011). The various advantages of producing pigments from
bacteria include ease of cultivation, colours of different shades, easy to manipulate
genes, structural complexicity suits for industrial needs, and growth on cheap
substrate with bulk production. Consequently natural pigments isolated from
microorganism inhabiting environments other than terrestrial are an attractive
research tool, not only for biochemist and microbiologist, but also for pharmaceutical
and clinicians. This is because the natural pigments not only have capacity to
increase the market acceptability, but also display various advantageous biological
activities which includes antioxidants and antimicrobial activities.
Despite thousand of marine bioactive compounds have been isolated and
identified previously, this study will focus on the pharmacologically active
pigmented compounds produced by marine bacteria. Even though pigmented
compounds produced by terrestrial samples are beyond the scope of the study, some
brief explanation and examples will still be mentioned for comparative purposes, in
order to outline common sources of natural pigments and its functions.
Among natural pigments, prodigiosin is an alkaloid group of pigments which
poses wide variety of biological activities which includes antibacterial, anticancer,
antimalarial, antiplasmodial, immunosuppressant, antifouling and many more
(Campas et al., 2003; Sertan-De-Guzman et al., 2007; Park et al., 2012). Hence, this
pigment is desired for many applications including for pharmaceutical and non-
pharmaceutical industries (such as aquaculture, cosmetics, food, textile etc). The
prodigiosin pigments, that poses antimicrobial potential is an additional advantages,
4
as the pigments can be simultaneously used as colouring agent and preservative
agent in many industrial applications. This is consequent with the increasing demand
for effective and non-toxic antibacterial therapeutics.
Colours are vital characteristics of textile products, where it is the first
characteristic perceived by the senses and aided in determining acceptability, judging
quality and increase basic aesthetic value of fabrics. Textile industry is one of the
rapidly growing industries world wide which utilizes enormous amounts of synthetic
dyes. In recent years, the demand of colorants from natural sources is alarming
worldwide due to the adverse impact of synthetics dyes. Moreover, the dyeing of
fabrics using prodigiosin exhibiting antibacterial agent have sponsored the
production of protective cloths especially to the hospitals fabrics, since the fabrics
has reported to be the vector of spreading many harmful bacteria from one person to
another person (Gupta & Laha, 2007).
5
1.3 Research objectives
The general objective of the present study was to focus on production of
natural pigments from a marine bacterium, and the pigments were evaluated on its
colouring and antimicrobial properties, which would be used as a colouring agent
and antimicrobial agent, simultaneously in textile industry. In order to achieve those,
there were specific objectives that had been planned for as the following:
1. To isolate pigmented bacteria from different marine environments and to
screen their pigment production as well their antimicrobial activity
2. To characterize and identify the potential strain of marine bacteria
3. To optimize the culture condition for growth, pigment production and
antimicrobial activity
4. To characterize and study the chemical profiling of the pigment
5. To evaluate the application of prodigiosin pigment from P. rubra BF1A
IBRL as colouring agent and antimicrobial agent in textile industry
1.4 Scope of study
This study was carried out to assess the ability marine bacteria in producing
pigment with antimicrobial activity which can be potential candidate for natural
coloring agent. The pigmented bacteria were isolated from marine samples, which
was then cultured and maintained in laboratory conditions for further studies. The
isolates were then screened for their potential in pigment production and antibacterial
activity. The screening for the bioactivity was done thoroughly where the solvent
effectiveness in extraction and partitioning was evaluated in order to select the
potential isolate with greater bioactivity. Screening for pigment production was done
6
using spectrophotometric analysis, whereas the screening for antimicrobial activity
was performed using disc diffusion assay and microdilution method. The potential
isolate was then characterized and identified based on its morphological, cultural,
microscopical, physiological and 16S rRNA molecular analysis. Next, several
physical parameter including light, pH, temperature, agitation rate and inoculum size
were optimized to improve the culture condition of the isolate for higher production
of pigment and antimicrobial activity. The chemical profiling of the pigment
produced was done using thin layer chromatography (TLC), preparative-TLC,
column chromatography, and high performance liquid chromatography (HPLC). The
toxicity level of the pigment was determined using brine shrimp (Artemia salina)
lethality test. In this analysis, the toxicity level of crude extract, partitioned and
fractionated extract was compared. The pigment extract exhibiting the antimicrobial
activity was then used to dye different types of fabric including wool, cotton, silk
satin and silk. The dyeability of the biopigment was evaluated using different types
of mordants. The colourfastness property of the naturally dyed fabric was then
evaluated towards light, washing, rubbing, perspiration and water. The antibacterial
property of the naturally dyed fabric was also determined.
7
CHAPTER 2: LITERATURE REVIEW
2.1 Definition of pigments and its classification
Pigments are colourful substances consisting of chromatophores, which either
absorb or reflect light in varying wavelengths of the visible region (Delgado-Vargas
et al., 2000). The absorbed light is dissipated in the pigment, and the reflected light is
visible as colours. The colours are also the result of a mix of residual wavelengths
that are reflected as stated by Mlodzinska (2009). There are different nature of
pigments which includes synthetic pigment, natural organic pigments, and also
natural inorganic pigments, which are classified based on their properties and origin.
Synthetic pigments (or artificial pigments) are organic pigments which
contain carbons and are chemically synthesized in laboratories. They have lower
toxicity effect compared to inorganic pigments and usually applied for colouring
agent for textiles, plastics, synthetic fibres, surface coating paints, inks and also
added to fish feed for pigmentation of the fishes (Ni et al., 2008). Examples of
synthetic pigments were diazo pigments, monoazo pigments, phthalocyanine
pigments, quinacridone pigments, astaxanthin and chantaxanthin (Mortensen, 2006).
Synthetic pigment is expensive, for example the price of synthetic astaxanthin is
approximately USD 2000 per kg (Ni et al., 2008). Nguyen (2013) stated that the
synthetic pigment cost for aquaculture industry is approximately 15 to 20% of the
total cost incurred and the potential market value for astaxanthin (carotenoid-type of
pigment) to be over 15 billion dollars for 2020.
On the other hand, natural inorganic pigments are mineral-earth pigment and
also can be termed as heavy metal pigments, which is derived mainly from minerals
such as gold and silver. Even though there are many naturally occurring pigments,
8
but the toxicity level of the natural pigments is always too high which finally limiting
its usage. Moreover, inorganic pigments commonly lack the intensity and brightness
of colour compared to organic pigments (Christie, 2001; Allam & Kumar, 2011).
Some examples of inorganic pigments are cobalt blue, chromium oxide, cadmium
yellow, molybdate orange and nickel titanate (Crisea & Vilarem, 2006).
Demands for pigments are increasing drastically due to the wide function to
industrial application and also to human health. The new awareness in human safety
and environmental conservation has kindled fresh enthusiasm for natural colorants.
The natural organic pigments are obtained from natural resources such as plants,
animals and microorganisms. The sources and distribution of natural pigments will
be discussed in detailed in Section 2.3. Natural pigments are also termed as bio-
pigments which are chemically and physically the most diverse group of pigments
(Salaudeen et al., 2010).
2.2 Disadvantages of synthetic pigment
The synthetic colorants have been used extensively in foods, medicines,
textile and cosmetics but the demand for the synthetic pigment has decreased through
the years due to its toxicological effect. The world production of colorants is 1
million tonnes per year (Christie, 2007) and it is estimated that over 0.7 million
tonnes of synthetic pigments are produced each year (Robinson et al., 2001). One of
the challenges of using synthetic pigment is the dependence on non-renewable oil
resources and sustainability (Venil et al., 2013).
9
2.2.1 Effect of synthetic pigments on environment
The majority of synthetic pigments are the azo derivatives (carcinogenic), and
thus the toxicity effect of the synthetic pigments is terrifying. The major
environmental problem of colorants is the removal of dyes and its intermediate
compounds from the effluent. Since the synthetic pigments represent large group of
organic chemicals, hence it is possible that such chemicals have undesirable effects
on environment and human health (Zollinger, 1987).
According to Delgado-Vargas et al. (2002), the synthesis of synthetic
pigments involves many types of intermediate organic compounds (including
aromatic and heterocyclic compounds) and also involves many reactions (nitration,
sulfonation, halogenations and amination). All the reaction involved in the
manufacturing processes are contributing to the release of the waste of the hazardous
organic compounds which lead to the environmental contamination when the waste
drained into the effluent. For example in textile industry, up to 200 000 tonnes of
synthetic pigments are lost to effluents every year during the dyeing and finishing
processes as a result of inefficient or incomplete dyeing processes (Zollinger, 1987).
Unfortunately, most of the dyes escape to the wastewater treatment plant and persist
in the environment as a result of high stability towards light, temperature, and
microbial attack.
Chemical toxicity in the environment also leads to the damage of aquatic
ecosystem, e.g soil fertility and aquatic organisms. The chemicals can affect the gas
solubility in water bodies and subsequently decrease photosynthetic activity in
aquatic life by reducing the light penetration (Banat et al., 1996). Moreover, the
chemicals can reduce the seed germination and plant growth, and inhibit the
10
elongation of shoot and roots of the plants associated with the untreated effluents
(Nirmalarani & Janardhanan, 1988).
2.2.2 Effect of synthetic pigments on human health
The effect of synthetic pigment towards human health can be categorised into
two types that are acute (short-term) and chronic (long-term) toxicological effect.
The acute toxicity involves oral ingestion and inhalation, skin irritation, skin
sensitisation and eye irritation (Christie, 2007). Whereas, the chronic toxicity of
synthetic pigment involves the genotoxicity effect when humans are exposed to the
synthetic pigments, which exhibits mutagenic, carcinogenic and teratogenics
characteristics (Christie, 2007). Some frequently used synthetic dyes that poses
negative effect towards human health are Reactive brilliant red, acid violet 7,
reactive black 5 and disperse blue 291 (Sudha et al., 2014).
The workers involved in the manufacturing of the synthetic dyes usually
become exposed to the dyes as well as the intermediate toxic chemicals used in their
manufacturing plant. Many years ago, it became apparent that workers involved in
the manufacturing of certain dyes, such as fuchsine, auramine, benzidine and 2-
naphthylamine had developed a high incident of bladder cancer. There is also
evidence that some reactive synthetic pigment caused contact dermatitis, allergic
conjunctivitis, rhinitis and occupational asthma to the workers dealing with the
synthetic pigments (Christie, 2007). The problem is caused by the ability of the
reactive dyes to combine with human serum albumin (HSA) to give dye-HSA
complex, which acts as an antigen. The antigen produces specific immunoglobulin E
11
(IgE) and cause allergic reactions through the release of chemicals such as histamine
(Luczynska & Topping, 1986).
A total of 50% of the world colorant production are the textile colorants. The
effects of synthetic textile colorant to human health are too obvious since its being
encountered in almost every aspect of our lives. For example, humans are in direct
contact with textile dyes because of the wearing cloths, and indirect contact with the
dyes because of the furnishing, such as carpets, curtains lounge suits, etc.
In terms of food colorant effect on human health, there are only few
permitted food colorants available (Delgado-Vargas et al., 2000), and indeed, both
permitted and non-permitted synthetic colorants are known to pose adverse effects.
Ashfaq & Masud (2002) stated that some non-permitted mutagenic and carcinogenic
food colours are still being used as food colorants, which includes auramine,
methanol yellow, lead chromate, orange-1 and malachite green. Tartrazine, a
permitted synthetic yellow colour that has been frequently used in sugar
confectioneries has been reported to be associated with irritability, restlessness and
sleep disturbance in hypertensive children (Rowe & Rowe, 1994).
2.3 Sources and distribution of natural pigments in nature
Pigments are distributed naturally in terrestrial and marine environment.
Marine and terrestrial sources differ from each other due to the influence of their
respective environmental conditions. In both terrestrial and marine environments, the
natural pigments are produced by biological sources which includes higher plants
(terrestrial), lower plants (marine) and microorganisms (both terrestrial and marine)
(Rivera & Canela-Garayoa, 2012; Ibrahim, 2008). Godinho & Boshle, 2008 divided
12
the biological materials containing pigments into two categories that are from
photosynthetic organisms (such as plants and some bacteria) and non-photosynthetic
organisms (such as bacteria, yeasts and fungi).
Humans and animals are not able to synthesize pigments de novo, hence they
need to acquire the pigment through their diet. Although animals do not synthesis the
pigments de novo, but they have been one of the pigment sources for human.
Animals that obtained pigments from their feed or diet, are either being metabolised
by the animal or the pigments accumulate inside their body especially in the skin.
Hence the animal still can be source of natural pigment for human, where the
pigment can act as an antioxidants and provitamin A to human health.
Plants and animal pigments are not suitable to be used as colouring agent for
industrial application because of the limited supply and vulnerable to damage of the
biodiversity (Shatila et al., 2013). Microorganisms are essential components of
earth’s biosphere (Whitman et al., 1998) which include fungi, yeasts and bacteria.
The study of pigment production from microorganisms has been started as early as
1926 (Snow & Fred, 1926) and now is one of the emerging fields of research.
Moreover, a number of research works have been reported over the last few years on
microbial pigments. Pigment is one of the secondary metabolite in microorganisms.
Microorganisms that are able to synthesis pigments are known as chromogenic
microbes.
2.3.1 Terrestrial environment (plants, animal, microbes)
2.3.1.1 Terrestrial plants
Plant pigmentation is among the oldest interest of botanist and plant was the
main source of pigments in 19th century (Christie, 2007). Plants can produce a
13
variety of pigments including indigo, chlorophylls, carotenoids, anthocyanins and
betalains (Davies, 2009; Mlodzinska, 2009). Generally, the parts of higher plants that
contain pigments are fruits (Susilowati, 2008; Nugraheni et al., 2010; Al-Sayed &
Kishk, 2011), vegetables, leaves (Biswas et al., 2013), roots and flowers as well (El-
Refai et al., 2010). Among the plant pigments, chlorophyll and carotenoid pigments
can be found in all terrestrial plants, which are also a major photosynthetic pigment.
This is because the primary function of pigments in plants is photosynthesis.
Chlorophyll is the green pigments that responsible for the green colours of the
leaves. Whereas, the variety of bright and attractive colours of fruits vegetables and
flowers are due to the carotenoid pigments (Corol et al., 2002). On the other hand,
the red, blue and purple coloured foliage are also caused by anthocyanin pigments
(Alkema & Seager, 1982), whereas the heartwood colour in certain plants is caused
by the quinine pigments (Delgado-Vargas et al., 2002). Table 2.1 lists some
pigments produced by plants in relative to its colour. The natural pigments from
plants have been extracted to be used for industrial application, for example in food
industry (Rymbal et al., 2011). Joshi et al. (2003) stated that most of plant natural
pigments have been extracted from grapes, paprika, and beet, previously.
2.3.1.2 Terrestrial animal
Very few studies have been reported about the distribution of natural organic
pigments in terrestrial animals. Amato et al. (2003) had studied the pigmentation
effect in terrestrial isopods, Atlantoscla floridana. Cochineal pigment from cochineal
insect (Dactylopius coccus costa) has been used since time immemorial in India,
Persia and Europe for colouring the clothes (Delgado-Vargas et al., 2002).
14
Table 2.1: Examples of pigment producing terrestrial plants
Pigments Types Example of
typical
colours
Plants References
Flavonoids Anthocyanin Blue, purple
and red
Beta vulgaris
Hibiscus sabdariffa
Solanum melongena
El-Refai et
al. (2010)
Carotenoids Carotenoids Yellow and
orange
Spinacia oleracea
Curcuma longa
Curcuma reticula
El-Refai et
al. (2010)
Cucuminoids Curcumin
Demethoxy-
curcumin
Bisdemethoxy-
curcumin
Yellow Curcuma longa Kulkarni et
al. (2012)
Betalains Amaranthin Red-violet Amaranthus tricolor Biswas et
al. (2013)
2.3.1.3 Terrestrial microorganisms
Terrestrial fungi are one of the significant microorganisms that are well
known in producing a wide range of pigments. This is because the fungal pigments
could be easily produced in high yields via cultivation technology (Mapari et al.,
2009; Qiu et al., 2010). There were over 1000 pigments being extracted from fungi
(Delgado-Vargas et al., 2002). Some of the fungi that are known to synthesize
pigments are Blakeslea trispora and Penicillium sp. (Gunasekaran & Poorniammal,
2008), Penicillium purpurogenum (Mendez et al., 2011) and Monodictys castaneae
(Visalalakchi & Muthumary, 2010). Among the natural pigments, β-carotene is the
major pigment produced by fungi. The other pigments produced by terrestrial fungi
are monascus pigment, rubropunctatin and monoscorubin (Joshi et al., 2003).
Besides, pigmented terrestrial yeasts are Phaffia sp. (Ni et al., 2006),
Sporobolomyces sp. (Maldonade et al., 2006 and Maldonade et al., 2007),
15
Rhodotorula sp. (Maldonade et al., 2006), Rhodotorula gaminis, Rhodotorula
glutinis, Rhodotorula mucilaginosa, Rhodotorula minuta, Rhodotorula roseus
(Maldonade et al., 2007,) and Rhodobacter (Chen et al., 2006). There are also studies
that mutate the yeasts to increase the yield of pigment production. Chew (2004) had
mutated the yeasts Xanthophyllomyces dendrorhous to produce carotenoids-type
pigment, which was later used to formulate fish feed. Some reporters has established
metabolic engineering of a non-chromogenic yeasts to produce pigment, as done by
Misawa & Shimada (1998).
Other terrestrial source of pigmented microorganisms is bacteria, which have
been isolated from different type of terrestrial samples. Most pigmented terrestrial
bacteria are isolated from soil samples. Lins et al. (2014), and Goswami et al. (2010)
had isolated bacteria from soil samples, which were capable in producing prodigiosin
and carotenoid pigments, respectively. The fact that pigmented bacteria were
predominant in soil was further confirmed by Indra Arulselvi et al. (2014), who had
isolated 24 of yellow pigmented bacteria from different types of soil under different
condition and climates. Similarly, Rashid et al. (2014) had isolated red, brown, pink,
black, blue, green, orange and yellow pigmented bacteria from 8 types of soil of
Dhaka City, Bangladesh.
Besides, many pigmented bacteria are also isolated from clinical samples. For
example, a clinical isolate of Pseudomonas aeruginosa, previously obtained from a
clinical laboratory was reported to produce pyocyanin pigments (Karpagam et al.,
2013). Serratia marcescens is another clinical isolate that has been isolated from
clinical sample (Samrot et al., 2011).
Moreover, Shatila et al. (2013) had isolated a bacterium, Exiguobacterium
aurantiacum FH from the air, and the bacterium is known to produce carotenoids-
16
type of pigment, whereas Serratia rubidaea was isolated from an agricultural farm,
which was known to produce pigment with antibacterial, antiproliferative and
immunosuppressive properties (Darshan, 2013). The terrestrial pigmented bacteria
also can be found in freshwater (Hardjito et al., 2002), and toilet water sources
(Gulani et al., 2012) confirming their widespread diversity.
2.3.2 Marine environment
Up to date, there are more than 20, 000 structurally diverse marine natural
compounds that have been isolated from the marine environment including the
pigmented compounds (Rocha-Martin et al., 2014) and there are hundreds of new
compounds that have been discovered every year from the marine environment
(Pabba et al., 2011). Surprisingly, the discovery rate of natural bioactive compounds
from marine microorganisms has surpasses that of the terrestrial counterparts
(Attimarad et al., 2012). However it is stated that the research into marine
environment is still in the early phase, and many mysteries associated with aquatic
fauna and flora have yet to be discovered (Soliev et al., 2011). In marine
environment the animals produced more bioactive compound compared to plants,
and this is in sharp contrast to the terrestrial environment, where the plants by far
exceeds animals in terms of production of natural bioactive substances (Proksch et
al., 2002).
The privileges and special condition of marine environment that differ from
other ecosystem is reflected by the physiology and biochemical properties of marine
organisms especially the microorganisms.
17
2.3.2.1 Marine plants (Algae)
In macroalgae, the pigments are stored in chloroplast and chromoplast
(Romero et al., 2012). Structures exhibited by the compounds produced by
macroalgae ranges from acyclic entities with a linear chain to complex polycyclic
molecules and included biogenic compounds such as terpenoids, phenolic
compounds, alkaloids, aldehydes, alcohols (Abad et al., 2011, Bagawathy et al.,
2011). Likewise the higher plants, the green colour of the macroalgae are also
contributed by chlorophyll pigment. On the other hand, the xanthophylls and
fucoxanthin pigments are responsible for the brown coloured algae, whereas
phycoerythrin and phycocyanine pigments contributed to the red colour
characteristics of algae (Abad et al., 2011).
2.3.2.2 Marine animal
Some sessile and non-sessile marine invertebrates are brilliantly coloured
especially the species inhabiting the shallow water (Banranayake, 2006) which are
also biological source of pigments. Donia & Hamann (2003) stated that the majority
of marine based bioactive compounds including pigments have been identified from
marine invertebrates especially the sponges (Hamid & Ahmad, 2013). Devi et al.
(2012) reported that one of the sessile invertebrates with brilliant coloration was sea-
anemones (Hetaractis magnifica and Stichodactyla haddoni), which the coloration is
due to the photosynthetic pigment of symbiotic zooxanthellae (a photosynthetic
algae) present in different tissues of the organisms (Bandaranayake, 2006).
Often, carotenoid types of pigments are incorporated into the animal feeds.
Steven (1948) had studied the carotenoids distribution in brown trout and found a
significant amount of carotene was stored in the liver and ovary of the fish.
18
Khanafari et al. (2007) had extracted a number of carotenoids compounds from
shrimp waste (comprising of head and carapace). Among the pigments, carotenoid-
types of pigment are the most valuable pigment for human health, where the pigment
acted as antioxidant (Dutta et al., 2005). Hence, animals including fish, crabs,
shrimps, and lobsters have been the pigment (carotenoids) sources for human health.
However, Rao & Rao (2007) stated that the main source of carotenoids for human is
from fruits and vegetables.
2.3.2.3 Marine microorganisms
Although the pigmented microorganisms is widely spread in terrestrial
nature, a number of microorganism distributed in marine environment is much higher
(Jensen & Fenical, 1994). Also, the marine ecosystem is presumed more
heterogeneous than the soil ecosystem at the bacterial level (Kim, 2013). Marine
microorganisms can be divided into three categories on the basis of habitat which are
psychrophiles (living at low temperature), halophiles (living at high salinity) and
barophiles (living under high pressure).
Marine microorganisms continue to be a major focus compared to other
marine macroorganism, with 10% increase in the number of compounds reported
from 2011 to 2012 (Blunt et al., 2004). It is estimated that marine oceans contains
the highest percentage of prokaryotic cells on earth that is 4-6 x 1030 cells (Whitman
et al., 1998), while in seawater, there are about 106 bacterial cells per millilitre
(Pabba et al., 2011).
There are five groups of marine microorganisms including fungi, yeast,
microalgae and bacteria. Marine fungi grow and sporulate exclusively in seawater,
19
and their spores are capable of germinating in seawater. Some of them are facultative
and there are over 800 obligate marine fungal strains reported so far. The fungus,
Monodictys sp. has been isolated from the surface of sea-urchin and is known to
produce antimicrobial anthraquinone pigment (El-Beih et al., 2007). Moreover,
marine yeasts, Aureobasidium pullulans had been isolated from marine sediment of
Southern Sea, China, which was able to produce an alkaline protease (Chi et al.,
2007).
Dunaliella sp., Haematococcus sp. (Phromthong et al., 2012), and Chlorella
vulgaris are some examples of the chromogenic microalgae (Gouveia & Empis,
2002) in marine environment. The production of pigment from Haematococcus
pluvialis has been developed in large scale in 1990 (Lorenz & Cysewski, 2000).
Marine microalgae such as diatoms and cyanobacteria are known to produce various
bioactive compounds including natural pigmented compounds and novel metabolites
(Abad et al., 2011), but have attracted little attention among researchers. Some
examples of the pigments produced by microalgae are β-carotene, asthaxanthin, and
canthaxanthin (Jissa, 2008; Guedes et al., 2011).
2.3.2.3.1 Marine bacteria
According to Jensen and Fenical, (1996), the general definition of marine
bacteria is “the microorganisms which are isolated from the marine habitat and
which are functionally reproductive under typical marine condition”.
The awareness of the role of marine microorganisms in biotechnology has
started over the past 30-40 years and the research regarding the marine
microorganisms is still growing where hundreds of new compounds are being
20
discovered every year (Proksch et al., 2002). However, at the end of 2008, there were
only 3000 microbial bioactive compounds had been reported from the marine
environment (Rahman, 2008). Hence, since the earth is covered by seas at
approximately 71% of its surface, and the microorganisms are distributed widely
from ocean shores to the deep sea floor, there are still many resources that crucially
need to be explored to isolate many more marine novel bioactive compounds.
One of the marine bacteria that is widely distributed in marine environment is
pigmented marine bacteria. Due to the enormous diversity of marine bacteria, it is
impossible to give a general number of pigmented bacteria to total of marine
heterotrophic bacteria. Marine pigmented bacteria have different mode of living,
which include free-living (referred to as pelagic or planctonic), attached to animate
or inanimate materials and also attached to internal space of invertebrates. However,
previous study stated that the bacteria with symbiotic relationship (attached to
animate resources) are likely to be advantageous to produce beneficial secondary
metabolites. The detailed explanation of symbiosis relationship is stated in section
2.3.2.3.1.
The important microhabitats for the prolific marine bacteria includes animate
(internal tissues and surface of invertebrates) and inanimate (sediments, stone
particles, seawater etc) resources. The marine microorganism exhibits a huge ability
to produce various kinds of bioactive compounds (secondary metabolites). Carte
(1996) stated that bioactive metabolites produced by marine bacteria have more
novel and striking structures compared to terrestrial microorganisms.
Pigment synthesis by marine bacteria is definitely dependent on the pH, light,
temperature of the nature, and the marine bacteria is capable in producing pigments
21
with almost all colours of the rainbow including black, white, brown, golden, silver,
florescent green, yellow or blue (Kim, 2013). Pigments produced by bacteria mostly
by the quorum sensing mechanism (Thomson et al., 2000; Slater et al., 2003).
Among the marine bacteria that are able to synthesize pigments are Bacillus
(Perez-Fons et al., 2011), Flavobacterium (Courington & Goodwin, 1955; Weeks
and Garner, 1967), Micrococcus (Sobin & Stahly, 1941; Courington & Goodwin,
1955; Kaiser et al., 2007; Ibrahim, 2008), Erwinia sp and Sarcina (Sobin & Stahly,
1941), Serratia (Teh Faridah, 2012), Pseudoalteromonas (Feher et al., 2008)
Pseudomonas (Angell et al., 2006) and Vibrio (Allihosseini et al., 2008). Table 2.2
shows the list of marine bacteria that have been reported to be able to produce
various pigmented bioactive compounds.
Table 2.2: Marine bacteria that were able to produce various pigmented
bioactive compounds
Marine bacteria Pigments Activity References
Pseudomonas
aeruginosa
Pyocyanin and
pyorubrin
Antibacterial Angell et al. (2006);
Saha et al. (2008)
Bacillus sp. Phenazine Cytotoxic Li et al. (2007)
Pseudoalteromonas
tunicata
Tambjamines Antibiotics,
anticancer
Franks et al. (2005);
Pinkerton et al. (2010)
Shewanella
colwelliana
Melanins Protection from
UV radiation
Fuqua & Weiner (1993);
Kotob et al. (2005)
Agrobacterium
auranticum
Astaxanthin Antioxidant Misawa et al. (2005)
Pseudoalteromonas
luteoviolacea,
Pseudoalteromonas
sp.
Violacein Antiprotozoan,
antibiotic,
anticancer
Novick & Teyler (1985);
Yada et al. (2008)
Serratia marcescens Anthocyanin Colouring agent Nerurkar et al. (2013)
Pseudomonas sp. Melanins Antioxidant Tarangini & Mishral
(2013)
Streptomyces
coelicolor,
S. violaceusruber,
S. lividans
Actinorhodin Indicator
compounds in
laboratory
Palanichamy et al.
(2011)
22
2.3.2.3.1.1 Advantageous of microorganisms as pigment sources over plants
and animals
Among microorganisms, bacteria have immense potential to produce various
bioactive compounds including the pigments. The single-celled organisms are the
most likely commercial source of pigments with biotechnological techniques as tools
for their exploitation (Delgado-Vargas et al., 2002). Practically, fermentation of
microorganisms could be a precious source of pigments but some have stated that the
pigment production via fermentation will generate cost (Maldonade et al., 2007).
However the cost can be reduced by cultivating the pigment producers in cheap
industrial by-products (e.g. agro-industrial residues) or waste products as nutrient
source or growth medium (Aksu & Eren, 2005) which will provide a profitable
means (Latha & Jeevarathnam, 2010).
Mata-Gomez et al. (2014) stated that industrial wastes such as chicken
feathers, whey, and crude glycerol have been used as substrates for yeasts cultivation
to produce carotenoids pigments (Valduga et al., 2009; Taskin et al., 2011; Saenge et
al., 2011). Further, Tarangini & Mishra (2013) also have used vegetable wastes to
cultivate a bacterium Pseudomonas sp. to produce melanin-type of pigment.
Venil et al. (2013) reported that microorganisms is mostly preferred
biological source of pigments compared to plants and animals owing to its ability to
produce more stable pigments, higher yields and also lower residues . Proksch et al.
(2001) also stated that the concentration of bioactive compounds present in the
marine invertebrates (macroorganisms) were very little, accounting for less than a
millionth of the wet weight. Likewise, Parthiban & Thilagavathi (2012) stated that
only per grams of pigment yielded from 1 kg of dried plant materials, causing the
23
current market price of plant pigments about US$ 1/g. The use of large amounts of
biomass of these invertebrates and plants, on the other hand will lead to extinction of
the respective species subsequently, damaging the ecosystem. Similarly, Maldonade
et al. (2007) stated that the natural pigment by plants may suffer from diminishing or
unstable supply of raw materials, subject to climate conditions, as well as varying
colorant level and subsequently affecting the quality of the final product.
There are facts stated that the bacteria produced pigments in very low
quantities, however the technologies to overcome this challenges is already in place,
which provide a route for introducing the bacterial pigments to a cost sensitive-
world. For example, the requirement for large number of Petri dishes for cultivating
certain bacteria can be overcome by using fermentation vessels. On the other hand,
the molecular approach can be used to clone the genes responsible for the
biosynthesis of pigments in order to increase the production of pigments from the
bacterial cells (Venil et al., 2013). The cloning and DNA recombinant technology
have been harnessed to overproduce the pigments since ancient time (Malpartida &
Hopwood, 1984).
The molecular approach also can be used to manipulate the biosynthetic
pathways of pigment production to engineer a structure of pigment and consequently
its colour. For example, Streptomyces coelicolor, which produced blue pigment
(actinorhodin) can be genetically modified to produce bright yellow pigment
(kalafungin), and alternatively actinorhodin pigment biosynthesis can be engineered
to produce orange or yellow-red anthraquinones pigment (Bartel et al., 1990;
McDaniel et al., 1993). The metabolic engineering of microorganisms for natural
pigment synthesis has been described in detailed by Venil et al. (2013).
24
On the other hand, microorganisms can be cultivated using various laboratory
medium which can be propagated easily compared to plant tissue culture. Therefore,
the microorganisms have been selected to as a sustainable resource for the biological
production of active compounds including the pigment.
2.3.2.3.1.2 Symbiosis
Marine plants and animals in marine environment are well known to have
developed relationship (beneficial interaction) with numerous microorganisms and
this association is known as epibiosis. The attaching microorganisms known as
ephibiotic, whereas the marine living organisms known as epibionts (Wahl, 1989).
Symbiosis is one of the common associations between the marine bacteria
(symbiotic) and its attaching host (simbionts) (Haygood et al., 1999).
According to Armstrong et al. (2001), the symbiotic bacteria living on the
surface of higher marine organisms could acquire the necessary nutrition from their
animal or plant host such as vitamins, polysaccharides and fatty acids. The bacteria
in return could excrete secondary products such as amino acid, antibiotics and toxin
propitious for the development, metabolism and also for chemical defence of the host
(Armstrong et al., 2001; Hamid et al., 2013). There are also studies reported that
marine symbiotic bacteria are important source of fixed nitrogen for the associated
algae (Goecke et al., 2010). The symbiotic bacteria that are able to protect its host
from settlement of other pathogenic microflora is known to poses antimicrobial
activity and will be great source for discovery of new drugs in pharmaceutical
industry and also can be great source of antifouling agent in aquaculture industry.